Wheat with increased resistant starch levels

ABSTRACT

A series of independent human-induced non-transgenic mutations found at one or more of the SBEII genes of wheat; wheat plants having these mutations in one or more of their SBEII genes; and a method of creating and finding similar and/or additional mutations of SBEII by screening pooled and/or individual wheat plants. The seeds and flour from the wheat plants of the present invention exhibit an increase in amylose and resistant starch without having the inclusion of foreign nucleic acids in their genomes. Additionally, the wheat plants of the present invention exhibit altered SBEII activity without having the inclusion of foreign nucleic acids in their genomes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 13/633,588 filed Oct. 2, 2012, which claims thebenefit of U.S. Provisional Application No. 61/542,953, entitled “Wheatwith increased resistant starch levels,” filed Oct. 4, 2011; theentirety of both applications is incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Some claims of this invention were made with government support underUnited States Department of Health and Human Services, NationalInstitute of Diabetes and Digestive and Kidney Diseases, grant numbers1R44DK085811-01A1, 4R44DK085811-02 and 5R44DK085811-03. The governmenthas certain rights in this invention.

FIELD

This invention relates to human-induced non-transgenic mutations in oneor more starch branching enzyme II (SBEII) genes. In one embodiment, theinvention relates to human-induced non-transgenic mutations in one ormore SBEII genes of wheat and wheat plants. In still another embodiment,human-induced non-transgenic mutations are in the SBEIIa and/or SBEIIbgene sequences, more particularly, combined mutations in SBEIIa and inboth SBEIIa and SBEIIb.

This invention further relates to wheat plants having wheat seeds andwheat flour with increased levels of amylose and increased levels ofresistant starch as a result of non-transgenic mutations in at least oneof their SBEII genes. This invention also relates to a method thatutilizes non-transgenic means to create wheat plants having mutations inat least one of their SBEII genes. In addition, this invention concernswheat flour and wheat-based food products made from the seeds of thesewheat plants having mutations in at least one of their SBEII genes.

BACKGROUND

An alarming number of adults and children in the United States areeither overweight or obese. Healthier food choices, including foods thatare high in resistant starch, can help people to better manage theirblood sugar levels and their weight. Resistant starch is defined asstarch that is not digested in the small intestine of healthyindividuals but is fermented in the large intestine. Due to its slowdigestion, resistant starch does not have the same caloric load asreadily digestible starch, nor does it cause as rapid a rise in bloodglucose levels after ingestion. Instead, resistant starch results in amore controlled glucose release over a longer period of time afterdigestion. This results in a decreased glycemic response, increasedinsulin sensitivity, and greater feelings of satiety. As a form ofdietary fiber, resistant starch contributes to better colon health dueto its fermentation by probiotic organisms in the lower gastrointestinaltract into short chain fatty acids, such as butyrate.

In the United States, the majority of dietary starch is consumed in theform of wheat based foods, such as bread, cereals, pastas, andtortillas, which contain very low levels of resistant starch. Cerealstarches typically contain less slowly digested amylose (about 25% oftotal starch) and more highly branched, rapidly digested amylopectin(about 75% of total starch). The amount of amylose in starch positivelycorrelates with the levels of dietary fiber and resistant starch. Incorn and barley, loss-of-function mutations of SBEIIb, one of severalenzymes in the starch synthesis pathway, have been identified. SBEIIb isthe predominant isoform of SBEII expressed in the endosperm of thesecrops and its loss results in increased amylose and resistant starchlevels. In contrast, both SBEIIa and SBEIIb are expressed in the wheatendosperm, but SBEIIa is the major isoform that is expressed in thiscrop. Though there has been great interest in finding mutations thatincrease amylose content (and therefore resistant starch content) inwheat, wheat lines with increased amylose levels are not commerciallyavailable. Preferred mutations would be single nucleotide polymorphisms(SNPs) that reduce or eliminate SBEII enzyme activity (and, in turn,increase amylose levels) without having significant negative pleiotropiceffects.

Identification of SNPs in wheat SBEII genes has proceeded slowlybecause, among other possible reasons, there is limited geneticdiversity in today's commercial wheat cultivars and bread wheat is apolyploid, with a complement of 7 chromosomes from each of threeancestors called the A, B and D genomes, resulting in a total of 21chromosomes. Typically, the bread wheat genome has three functionallyredundant copies of each gene (called homoeologs), and therefore, singlegene alterations usually do not produce any readily visible phenotypesuch as those that have been found in diploid corn. Often in wheat,altered variants of all three homoeologs must be combined genetically inorder to evaluate their effects. Pasta (durum) wheat is a tetraploid,consisting of A and B genomes, so only two altered copies of eachhomoeolog must be combined to obtain a phenotype.

To further compound these challenges, SBEIIa and SBEIIb are closelylocated on the same chromosome in wheat, making it difficult for allelesin these genes to be inherited independently unless through a rarerecombination event. Thus, it would be useful to have knock-down orknock-out mutations, resulting from SNPs, of both SBEIIa and SBEIIb ofeach genome of wheat. The availability of multiple allelic mutationswithin each SBEII locus, particularly within each SBEII locus of thesame genome, would allow for the breeding of new, non-geneticallymodified wheat lines with a spectrum of increased amylose and resistantstarch levels in seeds. Seeds from these lines could be used to producehealthier wheat-based food products, including flour, bread, cereals,pastas, and tortillas.

SUMMARY

In one embodiment, the invention relates to non-transgenic mutations inone or more SBEII genes. In one embodiment, one or more mutations are inthe SBEIIa gene. In another embodiment, one or more mutations are in theSBEIIb gene. In another embodiment, one or more mutations are in each ofthe SBEIIa and SBEIIb genes.

In one embodiment, the invention relates to multiple non-transgenicmutations in the SBEIIa gene including but not limited to 1, 2, 3, 4, 5,6, 7, 8, 9, 10, and greater than 10 mutations.

In another embodiment, the invention relates to multiple non-transgenicmutations in the SBEIIb gene including but not limited to 1, 2, 3, 4, 5,6, 7, 8, 9, 10, and greater than 10 mutations.

In another embodiment, the invention relates to multiple non-transgenicmutations in the SBEIIa gene including but not limited to 1, 2, 3, 4, 5,6, 7, 8, 9, 10, and greater than 10 mutations and multiple mutations inthe SBEIIb gene including but not limited to 1, 2, 3, 4, 5, 6, 7, 8, 9,10, and greater than 10 mutations.

In another embodiment, this invention relates to a wheat plant, wheatseeds, wheat plant parts, and progeny thereof with increased amylosecontent and increased resistant starch levels compared to wild typewheat plant, wheat seeds, wheat plant parts, and progeny thereof.

In another embodiment, this invention relates to a wheat plant, wheatseeds, wheat plant parts, and progeny thereof having reduced activity ofone or more SBEII enzymes compared to the wild type wheat plant, whereinthe reduction in SBEII enzyme activity is caused by a human-inducednon-transgenic mutation in one or more of the wheat plant's SBEII genes.In another embodiment, the SBEIIa enzyme has reduced activity. In yetanother embodiment, the SBEIIb enzyme has reduced activity. In stillanother embodiment, the SBEIIa and SBEIIb enzymes have reduced activity.

In another embodiment, this invention includes a wheat plant containingone or more mutated SBEII genes, as well as seeds, pollen, plant partsand progeny of that plant.

In another embodiment, this invention includes food and food productsincorporating wheat seeds and wheat flour having reduced SBEII enzymeactivity caused by a human-induced non-transgenic mutation in one ormore SBEII genes.

In another embodiment, this invention includes a wheat plant havingreduced activity of one or more SBEII enzymes compared to the wild typewheat plants, created by the steps of obtaining plant material from aparent wheat plant, inducing at least one mutation in at least one copyof an SBEII gene of the plant material by treating the plant materialwith a mutagen to create mutagenized plant material (e.g., seeds orpollen), analyzing progeny wheat plants to detect at least one mutationin at least one copy of a SBEII gene, selecting progeny wheat plantsthat have at least one mutation in at least one copy of an SBEII gene,crossing progeny wheat plants that have at least one mutation in atleast one copy of an SBEII gene with other progeny wheat plants thathave at least one mutation in a different copy of an SBEII gene, andrepeating the cycle of identifying progeny wheat plants having mutationsand crossing the progeny wheat plants having mutations with otherprogeny wheat plants having mutations to produce progeny wheat plantswith reduced SBEII enzyme activity. In another embodiment, the methodcomprises growing or using the mutagenized plant material to produceprogeny wheat plants.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO: 1 shows a partial Triticum aestivum gene for starch branchingenzyme IIa, A genome, exons 1-14.SEQ ID NO: 2 shows the partial protein sequence encoded by SEQ ID NO: 1.SEQ ID NO: 3 shows the Triticum aestivum SBEIIa gene for starchbranching enzyme IIa, B genome, exons 1-22 (GenBank Accession FM865435).SEQ ID NO: 4 shows the protein encoded by SEQ ID NO: 3 (GenBankAccession CAR95900).SEQ ID NO: 5 shows the Aegilops tauschii gene for starch branchingenzyme IIa, D genome, complete sequence exons 1-22 (GenBank AccessionAF338431).SEQ ID NO: 6 shows the protein encoded by SEQ ID NO: 5 (GenBankAccession AAK26821).SEQ ID NO: 7 shows a partial Triticum aestivum gene for starch branchingenzyme IIb, A genome, exons 1-11.SEQ ID NO: 8 shows the partial protein encoded by SEQ ID NO: 7.SEQ ID NO: 9 shows the partial Triticum aestivum gene for starchbranching enzyme IIb, B genome, exons 1-11.SEQ ID NO: 10 shows the partial protein encoded by SEQ ID NO: 9.SEQ ID NO: 11 shows the partial Aegilops tauschii gene for starchbranching enzyme IIb, D genome, exons 1-16 (GenBank Accession AY740398).SEQ ID NO: 12 shows the partial protein encoded by SEQ ID NO: 11(GenBank Accession AAW80632).SEQ ID NOs: 13-58 show exemplary homoeolog specific primers that haveproven useful in identifying useful mutations within the SBEIIa andSBEIIb gene sequences.

DETAILED DESCRIPTION Definitions

The numerical ranges in this disclosure are approximate, and thus mayinclude values outside of the range unless otherwise indicated.Numerical ranges include all values from and including the lower and theupper values, in increments of one unit, provided that there is aseparation of at least two units between any lower value and any highervalue. As an example, if a compositional, physical or other property,such as, for example, molecular weight, viscosity, etc., is from 100 to1,000, it is intended that all individual values, such as 100, 101, 102,etc., and sub ranges, such as 100 to 144, 155 to 170, 197 to 200, etc.,are expressly enumerated. For ranges containing values which are lessthan one or containing fractional numbers greater than one (e.g., 1.1,1.5, etc.), one unit is considered to be 0.0001, 0.001, 0.01 or 0.1, asappropriate. For ranges containing single digit numbers less than ten(e.g., 1 to 5), one unit is typically considered to be 0.1. These areonly examples of what is specifically intended, and all possiblecombinations of numerical values between the lowest value and thehighest value enumerated, are to be considered to be expressly stated inthis disclosure. Numerical ranges are provided within this disclosurefor, among other things, relative amounts of components in a mixture,and various temperature and other parameter ranges recited in themethods.

As used herein, the term “allele” is any of one or more alternativeforms of a gene, all of which relate to one trait or characteristic. Ina diploid cell or organism, the two alleles of a given gene occupycorresponding loci on a pair of homologous chromosomes.

As used herein, amino acid or nucleotide sequence “identity” and“similarity” are determined from an optimal global alignment between thetwo sequences being compared. An optimal global alignment is achievedusing, for example, the Needleman-Wunsch algorithm (Needleman andWunsch, 1970, J. Mol. Biol. 48:443-453). Sequences may also be alignedusing algorithms known in the art including but not limited to CLUSTAL Valgorithm or the Blastn or BLAST 2 sequence programs.

“Identity” means that an amino acid or nucleotide at a particularposition in a first polypeptide or polynucleotide is identical to acorresponding amino acid or nucleotide in a second polypeptide orpolynucleotide that is in an optimal global alignment with the firstpolypeptide or polynucleotide. In contrast to identity, “similarity”encompasses amino acids that are conservative substitutions. A“conservative” substitution is any substitution that has a positivescore in the Blosum62 substitution matrix (Hentikoff and Hentikoff,1992, Proc. Natl. Acad. Sci. USA 89: 10915-10919).

By the statement “sequence A is n % similar to sequence B,” it is meantthat n % of the positions of an optimal global alignment betweensequences A and B consists of identical residues or nucleotides andconservative substitutions. By the statement “sequence A is n %identical to sequence B,” it is meant that n % of the positions of anoptimal global alignment between sequences A and B consists of identicalresidues or nucleotides.

As used herein, the term “plant” includes reference to an immature ormature whole plant, including a plant from which seed or grain oranthers have been removed. A seed or embryo that will produce the plantis also considered to be the plant.

As used herein, the term “plant parts” includes plant protoplasts, plantcell tissue cultures from which wheat plants can be regenerated, plantcalli, plant clumps, and plant cells that are intact in plants or partsof plants, such as embryos, pollen, ovules, pericarp, seed, flowers,florets, heads, spikes, leaves, roots, root tips, anthers, and the like.

As used herein, the term “polypeptide(s)” refers to any peptide orprotein comprising two or more amino acids joined to each other bypeptide bonds or modified peptide bonds. “Polypeptide(s)” refers to bothshort chains, commonly referred to as peptides, oligopeptides andoligomers, and to longer chains generally referred to as proteins.Polypeptides may contain amino acids other than the 20 gene-encodedamino acids. “Polypeptide(s)” include those modified either by naturalprocesses, such as processing and other post-translationalmodifications, but also by chemical modification techniques. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature and they arewell known to those of skill in the art. It will be appreciated that thesame type of modification may be present in the same or varying degreeat several sites in a given polypeptide.

As used herein, an “SBEII derivative” refers to a SBEIIprotein/peptide/polypeptide sequence that possesses biological activitythat is substantially reduced as compared to the biological activity ofthe whole SBEII protein/peptide/polypeptide sequence. In other words, itrefers to a polypeptide of a modified SBEII protein of the inventionthat has reduced SBEII enzymatic activity. The term “SBEII derivative”encompasses the “fragments” or “chemical derivatives” of a modifiedSBEII protein/peptide.

As used herein, the term “polynucleotide(s)” generally refers to anypolyribonucleotide or poly-deoxyribonucleotide, which may be unmodifiedRNA or DNA or modified RNA or DNA. This definition includes, withoutlimitation, single- and double-stranded DNA, DNA that is a mixture ofsingle- and double-stranded regions or single-, double- andtriple-stranded regions, cDNA, single- and double-stranded RNA, and RNAthat is a mixture of single- and double-stranded regions, hybridmolecules comprising DNA and RNA that may be single-stranded or, moretypically, double-stranded, or triple-stranded regions, or a mixture ofsingle- and double-stranded regions. The term “polynucleotide(s)” alsoembraces short nucleotides or fragments, often referred to as“oligonucleotides,” that due to mutagenesis are not 100% identical butnevertheless code for the same amino acid sequence.

A “reduced or non-functional fragment,” as is used herein, refers to anucleic acid sequence that encodes for a SBEII protein that has reducedbiological activity as compared the protein coding of the whole nucleicacid sequence. In other words, it refers to a nucleic acid orfragment(s) thereof that substantially retains the capacity of encodingan SBEII polypeptide of the invention, but the encoded SBEII polypeptidehas reduced activity.

The term “fragment,” as used herein, refers to a polynucleotidesequence, (e.g, a PCR fragment) which is an isolated portion of thesubject nucleic acid constructed artificially (e.g., by chemicalsynthesis) or by cleaving a natural product into multiple pieces, usingrestriction endonucleases or mechanical shearing, or a portion of anucleic acid synthesized by PCR, DNA polymerase or any otherpolymerizing technique well known in the art, or expressed in a hostcell by recombinant nucleic acid technology well known to one of skillin the art.

With reference to polynucleotides of the invention, the term “isolatedpolynucleotide” is sometimes used. This term, when applied to DNA,refers to a DNA molecule that is separated from sequences with which itis immediately contiguous (in the 5′ and 3′ directions) in the naturallyoccurring genome of the organism from which it was derived. For example,the “isolated polynucleotide” may comprise a PCR fragment. In anotherembodiment, the “isolated polynucleotide” may comprise a DNA moleculeinserted into a vector, such as a plasmid or virus vector, or integratedinto the genomic DNA of a prokaryote or eukaryote. An “isolatedpolynucleotide molecule” may also comprise a cDNA molecule.

In one embodiment, the invention relates to non-transgenic mutations inone or more SBEII genes. In another embodiment, the invention describeswheat plants exhibiting seeds with increased amylose content andincreased resistant starch levels compared to wild type wheat seeds,without the inclusion of foreign nucleic acids in the wheat plants'genomes.

In still another embodiment, the invention relates to a series ofindependent human-induced non-transgenic mutations in one or more SBEIIgenes; wheat plants having one or more of these mutations in at leastone SBEII gene thereof; and a method of creating and identifying similarand/or additional mutations in at least one SBEII gene of wheat.Additionally, the invention relates to wheat plants exhibiting seed withincreased amylose and resistant starch content compared to wild typewheat seed, without the inclusion of foreign nucleic acids in theplants' genomes.

SBEII Mutations

A. SBEII Genes

In one embodiment, the invention relates to one or more non-transgenicmutations in the SBEII gene. In another embodiment, the SBEII gene maycontain one or more non-transgenic mutations recited in Tables 1-6 and8-12 and corresponding mutations in homoeologues and combinationsthereof.

In another embodiment, the invention comprises corresponding mutationsto the one or more non-transgenic mutations disclosed herein in theSBEII gene in a corresponding homoeologue. By way of example, anidentified mutation in the SBEIIa gene of the A genome may be abeneficial mutation in the SBEIIa gene of the B and/or D genome. One ofordinary skill in the art will understand that the mutation in thehomoeologue may not be in the exact location.

One of ordinary skill in the art understands there is natural variationin the genetic sequences of the SBEII genes in different wheatvarieties. The degree of sequence identity between homologous SBEIIagenes or the proteins is believed to be about 90%. This is true forSBEIIb genes and proteins as well.

The inventors have determined that to achieve a high amylose phenotypein wheat plants, mutations that reduce SBEII gene function aredesirable. Preferred mutations include missense and nonsense changes,including mutations that prematurely truncate the translation of one ormore SBEII proteins from messenger RNA, such as those mutations thatcreate a stop codon within the coding region of an SBEII messenger RNA.Such mutations include insertions, repeat sequences, splice junctionmutations, modified open reading frames (ORFs) and point mutations.

1. SBEIIa Genes

In another embodiment, the invention relates to one or more mutations inthe SBEIIa gene. In one embodiment, the invention relates to multiplenon-transgenic mutations in the SBEIIa gene including but not limited to1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and greater than 10 mutations.

In still another embodiment, one or more mutations are in the SBEIIagene of the A genome. In another embodiment, one or more mutations arein the SBEIIa gene of the B genome. In still another embodiment, one ormore mutations are in the SBEIIa gene of the D genome. In yet anotherembodiment, one or more mutations are in the SBEIIa genes of the A and Bgenomes. In still another embodiment, one or more mutations are in theSBEIIa genes of the A and D genomes. In another embodiment, one or moremutations are in the SBEIIa genes of the B and D genomes. In yet anotherembodiment, one or more mutations are in the SBEIIa genes of the A, B,and D genomes.

In one embodiment, one or more non-transgenic mutations are in bothalleles of the SBEIIa gene in the A genome. In another embodiment, thenon-transgenic mutations are identical in both alleles of the SBEIIagene of the A genome.

In one embodiment, one or more non-transgenic mutations are in bothalleles of the SBEIIa gene in the B genome. In another embodiment, thenon-transgenic mutations are identical in both alleles of the SBEIIagene of the B genome.

In one embodiment, one or more non-transgenic mutations are in bothalleles of the SBEIIa gene in the D genome. In another embodiment, thenon-transgenic mutations are identical in both alleles of the SBEIIagene of the D genome.

The following mutations are exemplary of the mutations created andidentified according to various embodiments of the invention. SEQ ID NOs1-6 are reference sequences for SBEIIa. SEQ ID NOs 7-12 are referencesequences for SBEIIb.

The following mutations identified in Tables 1-6 are exemplary of themutations created and identified according to various embodiments of theinvention. They are offered by way of illustration, not limitation. Itis to be understood that the mutations below are merely exemplary andthat similar mutations are also contemplated.

The nomenclature used in Tables 1-6 and 8-12 indicates the wild typenucleotide or amino acid, followed by its position according to thereferenced sequence, followed by the changed nucleotide or amino acid(A.A.) at that position using standard genetic code terminology. Anasterisk is used to designate a stop codon, also called a truncationmutation.

One exemplary mutation is G5267A, resulting in a change from guanine toadenine at nucleotide position 5267 identified according to its positionin the sequence of SEQ ID NO: 1. This mutation results in a change fromtryptophan to a stop mutation at amino acid position 436 identifiedaccording to its position in the expressed protein (SEQ ID NO: 2).

TABLE 1 Examples of mutations created and identified in SBEIIa in the Agenome of wheat plants. Nucleotide and amino acid changes are identifiedaccording to their positions in SEQ ID NOs: 1 and 2, respectively.Primer Nucleotide A.A. Variety SEQ IDs. Mutation Mutation PSSM SIFTExpress 13, 14 C538T V51= Express 13, 14 G586A E67= Express 13, 14 C605TP74S 0.89 Express 13, 14 G608A A75T 0.67 Express 13, 14 C644T IntronExpress 13, 14 G648A Intron Express 13, 14 C853T Intron Express 13, 14G951A G97= Express 13, 14 G952A G98R 0.44 Express 13, 14 G1036A E126K0.86 Express 13, 14 G1059A P133= Express 15, 16 C2384T Intron Express15, 16 C2384T Intron Express 15, 16 C2394T Intron Express 15, 16 G2574AIntron Express 15, 16 G2582A Splice Junction Express 15, 16 G2592A D260N10.4 0.3 Express 15, 16 G2605A G264D 22 0 Express 15, 16 G2612A K266=Express 15, 16 G2625A A271T 10.8 0.04 Express 15, 16 C2664T P284S 20.30.01 Express 15, 16 G2674A G287D 19.4 0 Express 15, 16 C2857T IntronExpress 15, 16 C2861T Intron Express 15, 16 C2921T Intron Express 15, 16G2990A E296K 0.03 Express 15, 16 C3004T F300= Express 15, 16 G3039AR312K 8.2 0.08 Express 15, 16 A3155T Intron Express 17, 18 C5164T IntronExpress 17, 18 C5164T Intron Express 17, 18 G5196A G413S 13.8 0 Kronos17, 18 G5239A G427D 6.6 0.09 Kronos 17, 18 C5256T H433Y 22.3 0 Express17, 18 G5267A W436* Kronos 17, 18 G5267A W436* Express 17, 18 G5268AD437N 7.9 0.04 Express 17, 18 G5268A D437N 7.9 0.04 Kronos 17, 18 G5268AD437N 7.9 0.04 Express 17, 18 G5289A G444R 19 0 Kronos 17, 18 G5289AG444R 19 0 Express 17, 18 G5298A E447K 8.9 0.02 Express 17, 18 G5301ASplice Junction Express 17, 18 G5301A Splice Junction Express 17, 18G5305A Intron Kronos 17, 18 G5308A Intron Express 17, 18 C5315T IntronExpress 17, 18 C5315T Intron Express 17, 18 C5315T Intron Express 17, 18C5324T Intron Kronos 17, 18 C5325T Intron Kronos 17, 18 G5332A IntronExpress 17, 18 G5386A Intron Express 17, 18 C5405T L453= Express 17, 18C5405T L453= Express 17, 18 G5418A R457K 18.3 0.01 Express 17, 18 G5422AW458* Kronos 17, 18 G5429A E461K 17.1 0.01 Kronos 17, 18 G5429A E461K17.1 0.01 Express 17, 18 G5432A E462K 17.6 0.01 Express 17, 18 G5432AE462K 17.6 0.01 Express 17, 18 G5448A G467E 27.1 0 Express 17, 18 G5463AG472E 27.1 0 Express 17, 18 G5463A G472E 27.1 0 Express 17, 18 G5463AG472E 27.1 0 Express 17, 18 G5464A G472= Express 17, 18 G5465A V473M17.1 0 Express 17, 18 C5470T T474= Kronos 17, 18 C5470T T474= Express17, 18 C5484T T479I 10.3 0.4 Kronos 17, 18 G5493A G482E 27.1 0 Kronos17, 18 G5522A Intron Express 17, 18 G5534A Intron Express 17, 18 G5655AIntron Express 17, 18 C5712T T488I 16.9 0 Express 17, 18 C5712T T488I16.9 0 Express 17, 18 C5719T N490= Express 17, 18 G5736A G496E 22.1 0Express 17, 18 C5745T T499I 15.8 0.02 Express 17, 18 G5753A D502N 17.10.01 Express 17, 18 G5756A A503T 19.8 0 Express 17, 18 C5757T A503V 19.20 Express 17, 18 G5783A D512N 7.8 0.18 Kronos 17, 18 C5801T H518Y −8.3 1Express 17, 18 C5804T P519S 26.7 0 Express 17, 18 C5811T A521V 6.3 0.21Express 17, 18 C5811T A521V 6.3 0.21 Express 17, 18 G5831A SpliceJunction Express 17, 18 G5852A Intron Express 17, 18 C5921T IntronExpress 17, 18 G5956A Intron Express 17, 18 G5956A Intron

In one embodiment, the invention relates to a polynucleotide of theSBEIIa gene in the A genome with one or more non-transgenic mutationslisted in Table 1 and corresponding to SEQ ID NO: 1. In anotherembodiment, the polynucleotide with one or more non-transgenic mutationslisted in Table 1 is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or greater than 99% identical to SEQ ID NO: 1.In yet another embodiment, the polynucleotide with one or morenon-transgenic mutations listed in Table 1 is 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than 99%similar to SEQ ID NO: 1.

In still another embodiment, the polynucleotide with one or morenon-transgenic mutation listed in Table 1 codes for a SBEIIa protein,wherein the SBEIIa protein comprises one or more non-transgenicmutations and is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or greater than 99% identical to SEQ ID NO: 2. Instill another embodiment, the polynucleotide with one or morenon-transgenic mutation listed in Table 1 codes for a SBEIIa protein,wherein the SBEIIa protein comprises one or more non-transgenicmutations and is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or greater than 99% similar to SEQ ID NO: 2.

Examples of mutations created and identified in SBEIIa in the B genomeof wheat plants are provided in Table 2. Nucleotide and amino acidchanges are identified according to their positions in SEQ ID NOs: 3 and4, respectively.

TABLE 2 Representative mutations in the SBEIIa gene in the B genomePrimer Nucleotide A.A. Variety SEQ IDs. Mutation Mutation PSSM SIFTExpress 23, 24 C4792T Intron Express 23, 24 G4830A Intron Express 23, 24C4878T Intron Kronos 23, 24 C4881T Intron Express 23, 24 C4937T IntronExpress 23, 24 C4960T T410I 4.8 0.25 Express 23, 24 C4960A T410N 13.90.02 Express 23, 24 C4961T T410= Express 23, 24 G4978A G416D 14.5 0.73Express 23, 24 G4987A G419D 16.8 0.01 Express 23, 24 G4987A G419D 16.80.01 Express 23, 24 C4990T T420I 21.4 0 Express 23, 24 C4998T H423Y 15.50.59 Express 23, 24 C5006T F425= Kronos 23, 24 G5011A G427D −0.4 0.5Express 23, 24 C5017T P429L 14.1 0.11 Express 23, 24 G5020A R430H 21.4 0Kronos 23, 24 G5020A R430H 21.4 0 Kronos 23, 24 G5020A R430H 21.4 0Kronos 23, 24 G5020A R430H 21.4 0 Kronos 23, 24 G5022A G431S 25.2 0Kronos 23, 24 C5025T H432Y −3.6 1 Express 23, 24 G5032A W434* Kronos 23,24 G5033A W434* Express 23, 24 G5036A M435I 15 0.03 Express 23, 24G5038A W436* Express 23, 24 G5038A W436* Kronos 23, 24 G5040A D437N 19.90.01 Express 23, 24 G5040A D437N 19.9 0.01 Express 23, 24 C5044T S438F12.1 0.01 Express 23, 24 G5062A G444E 17 0 Kronos 23, 24 G5062A G444E 170 Kronos 23, 24 G5062A G444E 17 0 Kronos 23, 24 G5063A G444= Kronos 23,24 G5065A S445N −4.7 1 Express 23, 24 G5068A W446* Express 23, 24 G5069AW446* Express 23, 24 G5069A W446* Kronos 23, 24 G5069A W446* Express 23,24 G5069A W446* Express 23, 24 G5069A W446* Express 23, 24 G5069A W446*Express 23, 24 G5070A E447K 9.3 0.02 Express 23, 24 G5070A E447K 9.30.02 Kronos 23, 24 G5073A Splice Junction Kronos 23, 24 G5080A IntronExpress 23, 24 C5081T Intron Express 23, 24 G5083A Intron Kronos 23, 24C5087T Intron Express 23, 24 C5090T Intron Kronos 23, 24 C5090T IntronKronos 23, 24 C5090T Intron Express 23, 24 C5090T Intron Express 23, 24G5092A Intron Kronos 23, 24 G5105A Intron Express 23, 24 G5112A IntronKronos 23, 24 G5112A Intron Kronos 23, 24 C5129T Intron Kronos 23, 24C5129T Intron Express 23, 24 C5158T Intron Express 23, 24 G5160A SpliceJunction Express 23, 24 G5161A V448I 0.01 Express 23, 24 G5161A V448I0.01 Express 23, 24 G5161A V448I 0.01 Express 23, 24 G5168A R450K 190.01 Express 23, 24 G5168A R450K 19 0.01 Kronos 23, 24 G5168A R450K 190.01 Express 23, 24 C5172T F451= Express 23, 24 G5185A A456T 13.3 0.11Express 23, 24 G5185A A456T 13.3 0.11 Kronos 23, 24 G5189A R457K 19 0.01Express 23, 24 G5193A W458* Express 23, 24 C5197T L460F 11.7 0.02Express 23, 24 G5200A E461K 18.3 0.01 Kronos 23, 24 G5203A E462K 18.3 0Express 23, 24 G5203A E462K 18.3 0 Kronos 23, 24 G5211A K464= Kronos 23,24 G5211A K464= Express 23, 24 G5219A G467E 27.7 0 Kronos 23, 24 G5219AG467E 27.7 0 Kronos 23, 24 G5219A G467E 21.1 0 Kronos 23, 24 G5219AG467E 27.7 0 Kronos 23, 24 T5223C F468= Express 23, 24 C5224T R469*Kronos 23, 24 G5233A G472R 27.3 0 Kronos 23, 24 G5234A G472E 27.7 0Kronos 23, 24 G5234A G472E 27.7 0 Express 23, 24 G5234A G472E 27.7 0Kronos 23, 24 C5240T T474I 21.9 0 Kronos 23, 24 C5244T S475= Express 23,24 C5255T T479I 9.8 0.55 Express 23, 24 G5264A G482E 27.7 0 Express 23,24 G5272A Splice Junction Express 23, 24 G5272A Splice Junction Kronos23, 24 G5272A Splice Junction Kronos 23, 24 G5276A Intron Express 23, 24G5284A Intron Express 23, 24 G5286A Intron Express 23, 24 G5287A IntronKronos 23, 24 G5287A Intron Kronos 23, 24 C5297T Intron Kronos 23, 24C5297T Intron Kronos 23, 24 G5306A Intron Express 23, 24 C5330T IntronExpress 23, 24 G5338A Intron Express 23, 24 G5350A Intron Express 23, 24G5350A Intron Express 23, 24 C5353T Intron Express 23, 24 G5364A IntronExpress 23, 24 G5364A Intron Express 23, 24 G5372A Intron Express 23, 24G5372A Intron Express 23, 24 C5379T Intron Express 23, 24 C5395T IntronExpress 23, 24 G5409A Intron Express 23, 24 G5421A Intron Express 23, 24C5448T Intron Express 23, 24 T5450C Intron Kronos 23, 24 C5469T IntronExpress 23, 24 G5472A Splice Junction Express 23, 24 G5475A M485I 0.18Express 23, 24 G5495A G492D −0.8 0.39 Express 23, 24 T5522A V501D 8.30.08 Express 23, 24 C5528A A503E 19.9 0 Express 23, 24 G5530A V504M 7.80.04 Express 23, 24 C5553T N511= Express 23, 24 G5566A G516R 5.2 0.32Express 23, 24 C5575T P519S 17.4 0.02 Kronos 23, 24 C5582T A521V 4.80.33 Kronos 23, 24 C5582T A521V 4.8 0.33 Express 23, 24 C5589T S523=Express 23, 24 G5606A Intron Express 23, 24 G5646A Intron Express 23, 24C5662T Intron Express 23, 24 C5662T Intron Express 23, 24 G5675A IntronExpress 23, 24 G5675A Intron Express 23, 24 G5835A Intron Express 23, 24C4960T T410I 4.8 0.25 Express 23, 24 G4987A G419D 16.8 0.01 Express 23,24 G5185A A456T 13.3 0.11 Express 23, 24 C5243T S475F 26.4 0 Express 23,24 C5255T T479I 9.8 0.55 Express 21, 22 G2386A G233D 0 Express 21, 22G2456A K256= Express 21, 22 G2464A Intron Express 21, 22 G2483A IntronExpress 21, 22 C2509T Intron Express 21, 22 C2518T Intron Express 21, 22G2606A A279T 3.1 0.14 Express 21, 22 C2610T P280L 5.1 0.47 Express 21,22 G2613A G281D 2.7 0.36 Express 21, 22 G2613A G281D 2.7 0.36 Express21, 22 C2648T P293S 0.08 Express 21, 22 G2661A Intron Express 21, 22G2661A Intron Express 21, 22 G2689A Intron Express 21, 22 G2945A SpliceJunction Express 21, 22 C2967T P303S 8.4 0.17 Express 21, 22 C2967TP303S 8.4 0.17 Express 21, 22 G2456A K256= Express 21, 22 C2518T IntronExpress 21, 22 G2606A A279T 3.1 0.14 Express 21, 22 G2606A A279T 3.10.14 Express 21, 22 C2648T P293S 0.08 Express 21, 22 G2661A IntronExpress 21, 22 C2967T P303S 8.4 0.17

In one embodiment, the invention relates to a polynucleotide of theSBEIIa gene in the B genome with one or more non-transgenic mutationslisted in Table 2 and corresponding to SEQ ID NO: 3. In anotherembodiment, the polynucleotide with one or more non-transgenic mutationslisted in Table 2 is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or greater than 99% identical to SEQ ID NO: 3.In yet another embodiment, the polynucleotide with one or morenon-transgenic mutations listed in Table 2 is 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than 99%similar to SEQ ID NO: 3.

In still another embodiment, the polynucleotide with one or morenon-transgenic mutation listed in Table 2 codes for a SBEIIa protein,wherein the SBEIIa protein comprises one or more non-transgenicmutations and is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or greater than 99% identical to SEQ ID NO: 4. Instill another embodiment, the polynucleotide with one or morenon-transgenic mutations listed in Table 2 codes for a SBEIIa protein,wherein the SBEIIa protein comprises one or more non-transgenicmutations and is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or greater than 99% similar to SEQ ID NO: 4.

Examples of mutations created and identified in SBEIIa in the D genomeof wheat plants are provided in Table 3. Nucleotide and amino acidchanges are identified according to their positions in SEQ ID NOs: 5 and6, respectively.

TABLE 3 Representative mutations in SBEIIa gene in the D genome PrimerNucleotide A.A. Variety SEQ IDs. Mutation Mutation PSSM SIFT Express 25,26 C1708T P60S 13.4 0.03 Express 25, 26 G1721A S64N −16.8 0.76 Express25, 26 G1753A E75K 0.74 Express 25, 26 G1753A E75K 0.74 Express 25, 26G1761A Q77= Express 25, 26 G1761A Q77= Express 25, 26 G1762A SpliceJunction Express 25, 26 G1762A Splice Junction Express 25, 26 G1780AIntron Express 25, 26 G1962A Intron Express 25, 26 G2037A SpliceJunction Express 25, 26 G1962A Intron Express 25, 26 G2037A SpliceJunction Express 25, 26 C1999T Intron Express 25, 26 G2185A E127K 0.79Express 25, 26 C1999T Intron Express 25, 26 C2011T Intron Express 25, 26C2028T Intron Express 25, 26 C2028T Intron Express 25, 26 C2032T IntronExpress 25, 26 G2065A A87T 0.59 Express 25, 26 G2065A A87T 0.59 Express25, 26 G2065A A87T 0.59 Express 25, 26 G2079A M91I 0.76 Express 25, 26G2086A G94R 0.15 Express 25, 26 G2087A G94E 0.43 Express 25, 26 G2126AG107D 0.53 Express 25, 26 G2131A V109M 0.14 Express 25, 26 G2134A E110K0.64 Express 25, 26 G2149A G115S 0.37 Express 25, 26 G2149A G115S 0.37Express 25, 26 G2183A G126E 1 Express 25, 26 G2187A E127= Express 25, 26G2220A G138= Express 25, 26 C2266T H154Y 16.9 0.03 Express 25, 26 C2286TIntron Express 25, 26 C2303T Intron Express 27, 28 C3589T S242= Express27, 28 C3602T H247Y 23.2 0 Express 27, 28 C3607A G248= Express 27, 28C3611G R250G 16 0.01 Express 27, 28 G3649A Intron Express 27, 28 G3677AIntron Express 27, 28 G3677A Intron Express 27, 28 C3743T S266F 16.9 0Express 27, 28 C3753T I269= Express 27, 28 C3772T P276S 9.5 0.35 Express27, 28 G3793A G283S 10.9 0.08 Express 27, 28 G3794A G283D 16.3 0.01Express 27, 28 G3824A Intron Express 27, 28 G4083A Intron Express 27, 28C4119T F296= Express 27, 28 C4126T P299S 9 0.15 Express 27, 28 C4127TP299L 18.1 0.01 Express 29, 30 G4818A E320K 7.9 0.11 Express 29, 30G4839A A327T 9.2 0.24 Express 29, 30 G4850A R330= Express 29, 30 G4850AR330= Express 29, 30 G4851A D331N 13 0.02 Express 29, 30 G4939A G360E24.5 0 Express 29, 30 C5118T Y361= Express 29, 30 G5144A S370N 22.9 0Express 29, 30 G5156A G374E 24.5 0 Express 29, 30 G5156A G374E 24.5 0Express 29, 30 G5166A E377= Express 29, 30 C5169T D378= Express 29, 30G5204A G390D 22.8 0 Express 29, 30 G5258A Intron Express 29, 30 C5267TIntron Express 29, 30 C5275T Intron Express 29, 30 G5299A Intron Express31, 32 G6793A A499T 18.7 0 Express 31, 32 C6163T Intron Express 31, 32G6793A A499T 18.7 0 Express 31, 32 C6163T Intron Express 31, 32 G6793AA499T 18.7 0 Express 31, 32 C6163T Intron Express 31, 32 G6174A IntronExpress 31, 32 C6183T Intron Express 31, 32 C6227T T406= Express 31, 32G6258A D417N 6.8 0.15 Express 31, 32 G6258A D417N 6.8 0.15 Express 31,32 C6275T H422= Express 31, 32 G6277A G423D 0.6 0.45 Express 31, 32G6277A G423D 0.6 0.45 Express 31, 32 G6286A R426H 21.5 0 Express 31, 32G6286A R426H 21.5 0 Express 31, 32 G6305A W432* Express 31, 32 G6306AD433N 20.1 0.01 Express 31, 32 G6306A D433N 20.1 0.01 Express 31, 32C6320T F437= Express 31, 32 G6327A G440R 17.2 0 Express 31, 32 G6328AG440E 17.3 0 Express 31, 32 G6329A G440= Express 31, 32 G6335A W442*Express 31, 32 G6336A E443K 9.4 0.02 Express 31, 32 C6418T IntronExpress 31, 32 G6426A Splice Junction Express 31, 32 C6442T L449=Express 31, 32 C6442T L449= Express 31, 32 G6451A A452T 13.2 0.08Express 31, 32 G6459A W454* Express 31, 32 C6463T L456F 11.6 0.02Express 31, 32 G6496A D467N 23.2 0 Express 31, 32 C6525T H476= Express31, 32 C6526T H477Y 21.5 0 Express 31, 32 G6538A Splice Junction Express31, 32 G6761A G488D −0.9 0.32 Express 31, 32 G6761A G488D −0.9 0.32Express 31, 32 G6793A A499T 18.7 0 Express 31, 32 G6796A V500I 5.8 0.15Express 31, 32 G6844A D516N 1.2 0.42 Express 31, 32 C6854T S519F 11.1 0Express 31, 32 G6860A G521D 15.5 0 Express 31, 32 G6860A G521D 15.5 0Express 31, 32 G6862A E522K 20.2 0 Express 31, 32 G6881A Intron Express31, 32 C6898T Intron

In one embodiment, the invention relates to a polynucleotide of theSBEIIa gene of the D genome with one or more non-transgenic mutationslisted in Table 3 and corresponding to SEQ ID NO: 5. In anotherembodiment, the polynucleotide with one or more non-transgenic mutationslisted in Table 3 is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or greater than 99% identical to SEQ ID NO: 5.In yet another embodiment, the polynucleotide with one or morenon-transgenic mutations listed in Table 3 is 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than 99%similar to SEQ ID NO: 5.

In still another embodiment, the polynucleotide with one or morenon-transgenic mutation listed in Table 3 codes for a SBEIIa protein,wherein the SBEIIa protein comprises one or more non-transgenicmutations and is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or greater than 99% identical to SEQ ID NO: 6. Instill another embodiment, the polynucleotide with one or morenon-transgenic mutation listed in Table 3 codes for a SBEIIa protein,wherein the SBEIIa protein comprises one or more non-transgenicmutations and is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or greater than 99% similar to SEQ ID NO: 6.

2. SBEIIb Genes

In another embodiment, one or more non-transgenic mutations are in theSBEIIb gene. In one embodiment, the invention relates to multiplenon-transgenic mutations in the SBEIIb gene including but not limited to1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and greater than 10 mutations.

In still another embodiment, one or more mutations are in the SBEIIbgene of the A genome. In another embodiment, one or more mutations arein the SBEIIb gene of the B genome. In still another embodiment, one ormore mutations are in the SBEIIb gene of the D genome. In yet anotherembodiment, one or more mutations are in the SBEIIb genes of the A and Bgenomes. In still another embodiment, one or more mutations are in theSBEIIb genes of the A and D genomes. In another embodiment, one or moremutations are in the SBEIIb genes of the B and D genomes. In yet anotherembodiment, one or more mutations are in the SBEIIb genes of the A, B,and D genomes.

In one embodiment, one or more non-transgenic mutations are in bothalleles of the SBEIIb gene in the A genome. In another embodiment, thenon-transgenic mutations are identical in both alleles of the SBEIIbgene of the A genome.

In one embodiment, one or more non-transgenic mutations are in bothalleles of the SBEIIb gene in the B genome. In another embodiment, thenon-transgenic mutations are identical in both alleles of the SBEIIbgene of the B genome.

In one embodiment, one or more non-transgenic mutations are in bothalleles of the SBEIIb gene in the D genome. In another embodiment, thenon-transgenic mutations are identical in both alleles of the SBEIIbgene of the D genome.

Examples of mutations created and identified in SBEIIb in the A genomeof wheat plants are provided in Table 4. Nucleotide and amino acidchanges are identified according to their positions in SEQ ID NOs: 7 and8, respectively.

TABLE 4 Representative Mutations in SBEIIb in the A genome PrimerNucleotide A.A. Variety SEQ IDs. Mutation Mutation PSSM SIFT Express 33,34 G211A Intron Express 33, 34 G278A W59* Express 33, 34 G298A G66D 6.10.03 Express 33, 34 G310A G70E 2.1 0.83 Express 33, 34 G310A G70E 2.10.83 Express 33, 34 C437T Intron Express 33, 34 G485A Intron Express 33,34 G547A V99I 0.84 Express 33, 34 G565A E105K 0.11 Express 33, 34 G678AT142= Express 33, 34 G680A G143E 1 Express 33, 34 G709A G153R 8.6 0.03Express 33, 34 C739T P163S 10.2 0.09 Express 33, 34 C743T T164M −3.40.21 Express 33, 34 G769A E173K −4.1 0.56 Express 35, 36 G1237A E201K16.7 0.21 Express 35, 36 C1307T Intron Express 35, 36 C1319T IntronExpress 35, 36 C1322T Intron Express 35, 36 G1341A G211S 14.9 0.02Express 35, 36 G1356A E216K 22.3 0 Express 35, 36 C1857T Intron Express37, 38 C2021T Intron Express 37, 38 C2021T Intron Express 35, 36 G2031AIntron Express 37, 38 C2072T Intron Express 37, 38 C2124T S259L 0.03Express 37, 38 C2126T P260S 0.23 Express 37, 38 G2142A G265D 3.6 0.17Express 37, 38 G2142A G265D 3.6 0.17 Express 37, 38 G2142A G265D 3.60.17 Express 37, 38 G2156A Splice Junction Express 37, 38 C2169T IntronExpress 37, 38 C2174T Intron Express 37, 38 G2244A G273S 0.6 0.31Express 37, 38 G2245A G273D −9.5 1 Express 37, 38 C2250T P275S 11.4 0.13Express 37, 38 G2282A W285* Express 37, 38 G2282A W285* Express 37, 38G2282A W285* Express 37, 38 C2293T S289F 8.4 0.02 Express 37, 38 C2340TP305S 15.8 0 Express 37, 38 C2344T P306L 17.3 0 Express 37, 38 C2344TP306L 17.3 0 Express 37, 38 G2349A E308K 0.07 Express 37, 38 A2441TIntron Express 37, 38 C2484T Intron Express 37, 38 G2525A Intron Express37, 38 G2535A E309K 0.03 Express 37, 38 G2540A K310= Express 37, 38C2556T P316S 11.5 0.07 Express 37, 38 C2606T G332= Express 37, 38 C2606TG332= Express 37, 38 C2617T P336L 18.2 0.01 Express 37, 38 C2642T IntronExpress 37, 38 G2697A Intron

In one embodiment, the invention relates to a polynucleotide of theSBEIIb gene of the A genome with one or more non-transgenic mutationslisted in Table 4 and corresponding to SEQ ID NO: 7. In anotherembodiment, the polynucleotide with one or more non-transgenic mutationslisted in Table 4 is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or greater than 99% identical to SEQ ID NO: 7.In yet another embodiment, the polynucleotide with one or morenon-transgenic mutations listed in Table 4 is 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than 99%similar to SEQ ID NO: 7.

In still another embodiment, the polynucleotide with one or morenon-transgenic mutation listed in Table 4 codes for a SBEIIb protein,wherein the SBEIIb protein comprises one or more non-transgenicmutations and is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or greater than 99% identical to SEQ ID NO: 8. Instill another embodiment, the polynucleotide with one or morenon-transgenic mutation listed in Table 4 codes for a SBEIIb protein,wherein the SBEIIb protein comprises one or more non-transgenicmutations and is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or greater than 99% similar to SEQ ID NO: 8.

Examples of mutations created and identified in SBEIIb in the B genomeof wheat plants are provided in Table 5. Nucleotide and amino acidchanges are identified according to their positions in SEQ ID NOs: 9 and10, respectively.

TABLE 5 Representative mutations in the SBEIIb gene in the B genomePrimer Nucleotide A.A. Variety SEQ IDs. Mutation Mutation PSSM SIFTExpress 41, 42 G371A G58R 0.26 Express 41, 42 C422T P75S 20.4 0.02Express 41, 42 G435A S79N 0.31 Express 41, 42 C1033T Intron Express 41,42 C1102T Intron Express 41, 42 C1102T Intron Express 41, 42 G1209AD129N 0.48 Express 41, 42 C1246T S141F 0.07 Express 41, 42 G1254A E144K0.91 Express 43, 44 G1916A S208N Express 43, 44 C2196T Intron Express43, 44 C2206T Intron Express 43, 44 G2221A A225T 6.9 0.21 Express 45, 46C2669T Intron Express 45, 46 C2776T P260S 10.4 0.21 Express 45, 46C2786T P263L 25.5 0.00 Express 45, 46 C2786T P263L 25.5 0.00 Express 45,46 C2919T S281L 9.9 0.09 Express 45, 46 C2786T P263L 25.5 0.00 Express45, 46 G3216A K319= Express 45, 46 C3232T R325W 27.3 0.00 Express 45, 46G3260A S334N 21.8 0.00 Express 47, 48 C3478T Intron Express 47, 48G3519A Intron Express 47, 48 G3678A Intron Express 47, 48 G3814A IntronExpress 47, 48 C3884T Intron Express 47, 48 C3993T L357F 8.5 0.11Express 47, 48 G4087A Intron Express 47, 48 C4419T Intron Express 47, 48G4280A Intron Express 47, 48 C4298T Intron Express 47, 48 C4374T IntronExpress 47, 48 C4374T Intron Express 47, 48 C4422T Intron Express 47, 48C4489T Intron

In one embodiment, the invention relates to a polynucleotide of theSBEIIb gene of the B genome with one or more non-transgenic mutationslisted in Table 5 and corresponding to SEQ ID NO: 9. In anotherembodiment, the polynucleotide with one or more non-transgenic mutationslisted in Table 5 is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or greater than 99% identical to SEQ ID NO: 9.In yet another embodiment, the polynucleotide with one or morenon-transgenic mutations listed in Table 5 is 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than 99%similar to SEQ ID NO: 9.

In still another embodiment, the polynucleotide with one or morenon-transgenic mutation listed in Table 5 codes for a SBEIIb protein,wherein the SBEIIb protein comprises one or more non-transgenicmutations and is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or greater than 99% identical to SEQ ID NO: 10. Instill another embodiment, the SBEIIb protein with one or morenon-transgenic mutations is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or greater than 99% similar to SEQ ID NO:10.

Examples of mutations created and identified in SBEIIb in the D genomeof wheat plants are provided in Table 6. Nucleotide and amino acidchanges are identified according to their positions in SEQ ID NOs: 11and 12, respectively.

TABLE 6 Representative mutations in SBEIIb in the D genome PrimerNucleotide A.A. Variety SEQ IDs. Mutation Mutation PSSM SIFT Express 49,50 G1691A G58E 0.76 Express 49, 50 C1742T P75L 17 0.01 Express 49, 50A1753G S79G 8.8 0.17 Express 49, 50 T1770C P84= Express 49, 50 C1784TP89L 0.28 Express 49, 50 C1831T Intron Express 49, 50 G1840A IntronExpress 49, 50 C1844T Intron Express 49, 50 C1844T Intron Express 49, 50C2438T Intron Express 49, 50 C2438T Intron Express 49, 50 C2463T IntronExpress 49, 50 C2479T P100S 0.32 Express 49, 50 T2511A D110E 0.98Express 49, 50 C2548T Q123* Express 49, 50 G2575A D132N 0.39 Express 49,50 G2649A Q156= Express 49, 50 C2672T T164M −5.3 0.46 Express 49, 50C2676T L165= Express 51, 52 C3142T Intron Express 51, 52 C3146T IntronExpress 51, 52 G3159A Intron Express 51, 52 G3185A R180K 1 Express 51,52 G3188A R181K 0.81 Express 51, 52 G3226A D194N 7 0.07 Express 51, 52G3226A D194N 7 0.07 Express 51, 52 G3226A D194N 7 0.07 Express 51, 52G3229A V195I 5.1 0.13 Express 51, 52 C3237T S197= Express 51, 52 C3246TY200= Express 51, 52 G3266A R207H 8.9 0.52 Express 51, 52 G3270A SpliceJunction Express 51, 52 C3279T Intron Express 51, 52 C3292T IntronExpress 51, 52 C3303T Intron Express 51, 52 C3318T Intron Express 51, 52C3330T Intron Express 51, 52 C3332T Intron Express 51, 52 G3345A A209T5.3 0.49 Express 51, 52 G3345A A209T 5.3 0.49 Express 51, 52 C3346TA209V 9.8 0.25 Express 51, 52 C3346T A209V 9.8 0.25 Express 51, 52C3346T A209V 9.8 0.25 Express 51, 52 G3364A R215Q 17.7 0.01 Express 51,52 C3410T Intron Express 51, 52 C3410T Intron Express 51, 52 C3416TIntron Express 51, 52 G3571A A224T 16.7 0.01 Express 51, 52 G3599A W233*Express 51, 52 G3628A Splice Junction Express 51, 52 C3662T IntronExpress 51, 52 C3662T Intron Express 53, 54 C4138T G265= Express 53, 54C4060T Intron Express 53, 54 G4080A G246D 0 Express 53, 54 C4124T P261S0.07 Express 53, 54 C4142T R267W 18 0 Express 53, 54 G4144A R267=Express 53, 54 C4159T Intron Express 53, 54 C4197A Intron Express 53, 54C4213T Intron Express 53, 54 G4229A Splice Junction Express 53, 54G4229A Splice Junction Express 53, 54 C4246T P275L 16.1 0.05 Express 53,54 C4246T P275L 16.1 0.05 Express 53, 54 G4260A D280N 15.8 0.07 Express53, 54 C4280T I286= Express 53, 54 G4290A V290M 13.3 0.01 Express 53, 54C4299T P293S 8.1 0.29 Express 53, 54 G4303A G294E 4 0.25 Express 53, 54C4311T P297S 17.3 0.07 Express 53, 54 G4347A Splice Junction Express 53,54 C4361T Intron Express 53, 54 G4515A Intron Express 53, 54 C4546TP316S 9.2 0.13 Express 53, 54 C4546T P316S 9.2 0.13 Express 53, 54C4546T P316S 9.2 0.13 Express 53, 54 C4546T P316S 9.2 0.13 Express 53,54 C4547T P316L 18.1 0.01 Express 53, 54 C4573T R325W 22.1 0 Express 53,54 C4605T S335= Express 53, 54 G4609A Splice Junction Express 53, 54G4609A Splice Junction Express 53, 54 C4618T Intron Express 57, 58C7427T D425= Express 57, 58 C7450T T433M 12.8 0 Express 57, 58 G7471AG440D 2.1 0.26 Express 57, 58 C7488T H446Y 23.3 0 Express 57, 58 C7506TR452C 25.4 0 Express 57, 58 C7506T R452C 25.4 0 Express 57, 58 G7537AIntron Express 57, 58 C7597T Intron Express 57, 58 G7635A R463= Express57, 58 G7655A R470K 13.6 0.05 Express 57, 58 G7669A E475K 17.2 0 Express57, 58 G7685A G480D 26 0 Express 57, 58 C7689T F481= Express 57, 58G7700A G485D 26 0 Express 57, 58 G7702A A486T 5.3 0 Express 57, 58C7758T Intron Express 57, 58 C7886T Intron Express 57, 58 G7897A V498I0.13 Express 57, 58 C7917T Y504= Express 57, 58 C7952T A516V 18.5 0Express 57, 58 G7968A M521I 18.9 0 Express 57, 58 G8056A Intron

In one embodiment, the invention relates to a polynucleotide of theSBEIIb gene of the D genome with one or more non-transgenic mutationslisted in Table 6 and corresponding to SEQ ID NO: 11. In anotherembodiment, the polynucleotide with one or more non-transgenic mutationslisted in Table 6 is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or greater than 99% identical to SEQ ID NO: 11.In yet another embodiment, the polynucleotide with one or morenon-transgenic mutations listed in Table 6 is 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than 99%similar to SEQ ID NO: 11.

In still another embodiment, the polynucleotide with one or morenon-transgenic mutation listed in Table 6 codes for a SBEIIb protein,wherein the SBEIIb protein comprises one or more non-transgenicmutations and is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or greater than 99% identical to SEQ ID NO: 12. Instill another embodiment, the SBEIIb protein with one or morenon-transgenic mutations is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or greater than 99% similar to SEQ ID NO:12.

3. Mutations in Both SBEIIa and SBEIIb Genes

In one embodiment, the invention relates to multiple non-transgenicmutations in the SBEIIa gene including but not limited to 1, 2, 3, 4, 5,6, 7, 8, 9, 10, and greater than 10 mutations and multiplenon-transgenic mutations in the SBEIIb gene including but not limited to1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and greater than 10 mutations.

In still another embodiment, one or more mutations are in each of theSBEIIa and SBEIIb genes of the A genome. In one embodiment, theinvention relates to multiple non-transgenic mutations in the SBEIIagene including but not limited to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, andgreater than 10 mutations and multiple non-transgenic mutations in theSBEIIb gene including but not limited to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,and greater than 10 mutations.

In another embodiment, one or more mutations are in each of the SBEIIaand SBEIIb genes of the B genome. In still another embodiment, one ormore mutations are in each of the SBEIIa and SBEIIb genes of the Dgenome. In yet another embodiment, one or more mutations are in each ofthe SBEIIa and SBEIIb genes of the A and B genomes. In still anotherembodiment, one or more mutations are in each of the SBEIIa and SBEIIbgenes of the A and D genomes. In another embodiment, one or moremutations are in each of the SBEIIa and SBEIIb genes of the B and Dgenomes. In yet another embodiment, one or more mutations are in each ofthe SBEIIa and SBEIIb genes of the A, B, and D genomes. In yet anotherembodiment, one or more mutations are in each of the SBEIIa genes of theA, B, and D genomes and additional mutations are in more or more of theSBEIIb genes of the A, B, and D genomes.

B. SBEII Proteins

Starch is a mixture of amylose and amylopectin, both of which are Glcpolymers. Amylose is a mostly linear polymer of 200 to 2000 α-1,4-bondedGlc moieties with rare α-1,6 branch points (for reviews, see Martin andSmith, 1995; Ball et al., 1996). Amylopectin is highly α-1,6-branched,with a complex structure of 10⁶ to 10⁸ M_(r) and up to 3×10⁶ Glcsubunits, making it one of the largest biological molecules in nature.

In the plant, starch is deposited as starch granules in chloroplasts ofphotosynthetic tissues or in amyloplasts of endosperm, embryos, tubers,and roots. In most plants, starch consists of 20% to 30% amylose and 70%to 80% amylopectin. In photosynthetic and nonphotosynthetic tissues theGlc moiety of ADP-Glc is incorporated in the growing amylose polymerwith the help of starch synthases. The formation of α-1,6 linkages inamylopectin is catalyzed by SBEs.

In yet another embodiment, the invention relates to one or morenon-transgenic mutations in the SBEII gene (as discussed above in thesection entitled SBEII Mutations) that result in an SBEII protein withone or more mutations as compared to wild type SBEII protein. In oneembodiment, the non-transgenic mutations include but are not limited tothe mutations recited in Tables 1-6 and 8-12, corresponding mutations inhomoeologues, and combinations thereof.

In another embodiment, the invention relates to one or morenon-transgenic mutations in the SBEII gene that inhibits production ofthe SBEII protein. In some embodiments, a mutation in the SBEII geneinhibits expression of the SBEII protein. In other embodiments, amutation in the SBEII gene creates an unstable or reduced function SBEIIprotein.

In another embodiment, the expression level of SBEII protein with one ormore mutations disclosed herein is reduced to 0-2%, 2-5%, 5-7%, 7-10%,10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, 45-50%, 50-60%,60-70%, 70-80%, 80-90%, 90-95%, and 95-99% of the expression level ofthe wild type SBEII protein.

In yet another embodiment, the expression level of SBEIIa protein withone or more mutations disclosed herein is reduced to 0-2%, 2-5%, 5-7%,7-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, 45-50%,50-60%, 60-70%, 70-80%, 80-90%, 90-95%, and 95-99% of the expressionlevel of the wild type SBEIIa protein.

In still another embodiment, the expression level of SBEIIb protein withone or more mutations disclosed herein is reduced to 0-2%, 2-5%, 5-7%,7-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, 45-50%,50-60%, 60-70%, 70-80%, 80-90%, 90-95%, and 95-99% of the expressionlevel of the wild type SBEIIb protein.

In yet another embodiment, the activity of the SBEII protein with one ormore mutations disclosed herein is reduced to 0-1, 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 69, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 86, 97,98, 99% and greater than 99% of the activity level of the wild typeSBEII protein. In another embodiment, the SBEII protein with one or moremutations disclosed herein has no activity or zero activity as comparedto wild type SBEII protein.

In still another embodiment, the activity of the SBEIIa protein with oneor more mutations disclosed herein is reduced to 0-1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 69, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 86, 97,98, 99% and greater than 99% of the activity level of the wild typeSBEIIa protein. In another embodiment, the SBEIIa protein with one ormore mutations disclosed herein has no activity or zero activity ascompared to wild type SBEIIa protein.

In yet another embodiment, the activity of the SBEIIb protein with oneor more mutations disclosed herein is reduced to 0-1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 69, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 86, 97,98, 99% and greater than 99% of the activity level of the wild typeSBEIIb protein. In another embodiment, the SBEIIb protein with one ormore mutations disclosed herein has no activity or zero activity ascompared to wild type SBEIIb protein.

C. Wheat Cultivars

In one embodiment, a wheat cultivar having at least one SBEII gene thatis diploid, polyploid, tertraploid, and hexaploid may be used.

In another embodiment, the wheat is Triticum aestivum.

In one embodiment, any cultivar of wheat can be used to create mutationsin an SBEII gene. In one embodiment, any cultivar of wheat can be usedto create mutations in an SBEIIa gene. In another embodiment, anycultivar of wheat can be used to create mutations in an SBEIIb gene.

In one embodiment, any cultivar of wheat can be used as lines to crossSBEII mutations into different cultivars. In still another embodiment,any cultivar of wheat can be used as lines to cross SBEIIa mutationsinto different cultivars. In another embodiment, any cultivar of wheatcan be used as lines to cross SBEIIb mutations into different cultivars.

In another embodiment, any cultivar of wheat having at least one SBEIIgene may be used including but not limited to hard red spring wheat,hard white wheat, durum wheat, soft white spring wheat, soft whitewinter wheat, hard red winter wheat, common wheat, spelt wheat, emmerwheat, pasta wheat and turgidum wheat.

In one embodiment, hard red spring wheat includes but is not limited toBullseye, Cabernet, Cal Rojo, Hank, Joaquin, Kelse, Lariat, Lassik,Malbec, Mika, PR 1404, Redwing, Summit 515, SY 314, Triple IV, Ultra,WB-Patron, WB-Rockland, Yecora Rojo, Accord, Aim, Anza, Baker, BethHashita, Bonus, Borah, Brim, Brooks, Buck Pronto, Butte 86, Cavalier,Challenger, Chief, Ciano T79, Colusa, Companion, Copper, Cuyama, Dash12, Eldon, Enano, Express, Expresso, Jefferson, Genero F81, Grandin,Helena 554, Hollis, Imuris T79, Inia 66R, Jerome, Kern, Len, Marshall,McKay, Nomad, Northwest 10, Oslo, Pavon F76, Pegasus, Pitic 62, PocoRed, Powell, Probrand 711, Probrand 751, Probrand 771, Probrand 775,Probred, Prointa Queguay, Prointa Quintal, Rich, RSI 5, Sagittario,Scarlet, Serra, Shasta, Solano, Spillman, Sprite, Stander, Stellar,Stoa, Success, Summit, Sunstar 2, Sunstar King, Tadinia, Tammy, Tanori71, Tara 2000, Tempo, Tesia T79, Topic, UI Winchester, Vance, Vandal,W444, Wampum, Wared, WB-Fuzion, Westbred 906R, Westbred 911, Westbred926, Westbred 936, Westbred Discovery, Westbred Rambo, Yolo, and Zeke.

In another embodiment, hard white wheat includes but is not limited toBlanca Fuerte, Blanca Grande 515, Blanca Royale, Clear White, Patwin,Patwin 515, WB-Cristallo, WB-Paloma, WB-Perla, Alta Blanca, BlancaGrande, Delano, Golden Spike, ID377S, Klasic, Lochsa, Lolo, Macon, Otis,Phoenix, Pima 77, Plata, Pristine, Ramona 50, Siete Cerros 66, Vaiolet,and Winsome.

In yet another embodiment, durum wheat includes but is not limited toCrown, Desert King, Desert King HP, Duraking, Fortissimo, Havasu,Kronos, Maestrale, Normanno, Orita, Platinum, Q-Max, RSI 59, Saragolla,Tango, Tipai, Topper, Utopia, Volante, WB-Mead, Westmore, Aldente,Aldura, Altar 84, Aruba, Bittern, Bravadur, Candura, Cortez, Deluxe,Desert Titan, Durex, Durfort, Eddie, Germains 5003D, Imperial, Kofa,Levante, Matt, Mead, Mexicali 75, Minos, Modoc, Mohawk, Nudura,Ocotillo, Produra, Reva, Ria, Septre, Sky, Tacna, Titan, Trump, Ward,Westbred 803, Westbred 881, Westbred 883, Westbred 1000D, WestbredLaker, Westbred Turbo, and Yavaros 79.

In another embodiment, soft white spring wheat includes but is notlimited to Alpowa, Alturas, Babe, Diva, JD, New Dirkwin, Nick, Twin,Whit, Blanca, Bliss, Calorwa, Centennial, Challis, Dirkwin, Eden,Edwall, Fielder, Fieldwin, Jubilee, Louise, Owens, Penawawa, Pomerelle,Sterling, Sunstar Promise, Super Dirkwin, Treasure, UI Cataldo, UIPettit, Urquie, Vanna, Waduel, Waduel 94, Wakanz, Walladay, Wawawai,Whitebird, and Zak.

In still another embodiment, soft white winter wheat includes but is notlimited to AP Badger, AP Legacy, Brundage 96, Bruneau, Cara, Goetze,Legion, Mary, Skiles, Stephens, SY Ovation, Tubbs, WB-Junction, WB-528,Xerpha, Yamhill, Barbee, Basin, Bitterroot, Bruehl, Castan, Chukar,Coda, Daws, Edwin, Eltan, Faro, Finch, Foote, Gene, Hill 81, Hiller,Hubbard, Hyak, Hyslop, Idaho 587, Kmor, Lambert, Lewjain, MacVicar,Madsen, Malcolm, Masami, McDermid, Moro, Nugaines, ORCF-101, ORCF-102,ORCF-103, Rod, Rohde, Rulo, Simon, Salute, Temple, Tres, Tubbs 06,UICF-Brundage, WB-523, and Weatherford.

In another embodiment, hard red winter wheat includes but is not limitedto Andrews, Archer, Batum, Blizzard, Bonneville, Boundary, Declo,Deloris, Finley, Garland, Hatton, Hoff, Longhorn, Manning, Meridian,Promontory, Vona, Wanser, Winridge.

In another embodiment, common wheat (hexaploid, free threshing),Triticum aestivum ssp aestivum includes but is not limited to Sonora,Wit Wolkoring, Chiddam Blanc De Mars, India-Jammu, Foisy.

In still another embodiment, spelt wheat (hexaploid, not freethreshing), Triticum aestivum ssp spelta includes but is not limited toSpanish Spelt, Swiss Spelt.

In yet another embodiment, Emmer Wheat (tetraploid), Triticum turgidumssp. dicoccum includes but is not limited to Ethiopian Blue Tinge.

In another embodiment, pasta wheat (tetraploid, free threshing),Triticum turgidum ssp durum includes but is not limited to Blue Beard,Durum-Iraq.

In yet another embodiment, Turgidum Wheat (tetraploid, free threshing),Triticum turgidum ssp turgidum includes but is not limited to Akmolinka,Maparcha.

In one embodiment, a cultivar of wheat having at least one SBEII genewith substantial percent identity to SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11 may be used in theinvention.

As used herein with regard to the wheat cultivars, “substantial percentidentity” means that the DNA sequence of the gene is sufficientlysimilar to SEQ ID NO: 1, 3, 5, 7, 9, or 11 at the nucleotide level tocode for a substantially similar protein, allowing for allelicdifferences (or alternate mRNA splicing) between cultivars. Inaccordance with one embodiment of the invention, “substantial percentidentity” may be present when the percent identity in the coding regionbetween the SBEII gene and SEQ ID NO: 1, 3, 5, 7, 9, or 11 is as low asabout 85%, provided that the percent identity in the conserved regionsof the gene is higher (e.g., at least about 90%). Preferably the percentidentity in the coding region is 85-90%, more preferably 90-95%, andoptimally, it is above 95%. Thus, one of skill in the art may prefer toutilize a wheat cultivar having commercial popularity or one havingspecific desired characteristics in which to create the SBEII-mutatedwheat plants, without deviating from the scope and intent of the presentinvention. Alternatively, one of skill in the art may prefer to utilizea wheat cultivar having few polymorphisms, such as an in-bred cultivar,in order to facilitate screening for mutations within one or more SBEIIgenes in accordance with the present invention.

Representative Methodology for Identification of SBEII Mutations

In order to create and identify the SBEII mutations and wheat plants ofthe invention, a method known as TILLING was utilized. See McCallum etal., Nature Biotechnology 18:455-457, 2000; McCallum et al., PlantPhysiology, 123:439-442, 2000; U.S. Publication No. 20040053236; andU.S. Pat. No. 5,994,075, all of which are incorporated herein byreference. In the basic TILLING methodology, plant materials, such asseeds, are subjected to chemical mutagenesis, which creates a series ofmutations within the genomes of the seeds' cells. The mutagenized seedsare grown into adult M1 plants and self-pollinated. DNA samples from theresulting M2 plants are pooled and are then screened for mutations in agene of interest. Once a mutation is identified in a gene of interest,the seeds of the M2 plant carrying that mutation are grown into adult M3plants and screened for the phenotypic characteristics associated withthe gene of interest.

The hexaploid cultivar Express and the tetraploid cultivar Kronos wereused.

In one embodiment, seeds from wheat are mutagenized and then grown intoM1 plants. The M1 plants are then allowed to self-pollinate and seedsfrom the M1 plant are grown into M2 plants, which are then screened formutations in their SBEII loci. While M1 plants can be screened formutations in accordance with alternative embodiments of the invention,one advantage of screening the M2 plants is that all somatic mutationscorrespond to germline mutations.

One of skill in the art will understand that a variety of wheat plantmaterials, including, but not limited to, seeds, pollen, plant tissue orplant cells, may be mutagenized in order to create the SBEII-mutatedwheat plants of the invention. However, the type of plant materialmutagenized may affect when the plant DNA is screened for mutations. Forexample, when pollen is subjected to mutagenesis prior to pollination ofa non-mutagenized plant, the seeds resulting from that pollination aregrown into M1 plants. Every cell of the M1 plants will contain mutationscreated in the pollen, thus these M1 plants may then be screened forSBEII mutations instead of waiting until the M2 generation.

Mutagens that create primarily point mutations and short deletions(about 1 to about 30 nucleotides), insertions, transversions, and ortransitions, such as chemical mutagens or radiation, may be used tocreate the mutations. Mutagens conforming with the method of theinvention include, but are not limited to, ethyl methanesulfonate (EMS),methylmethane sulfonate (MMS), N-ethyl-N-nitrosourea (ENU),triethylmelamine (TEM), N-methyl-N-nitrosourea (MNU), procarbazine,chlorambucil, cyclophosphamide, diethyl sulfate, acrylamide monomer,melphalan, nitrogen mustard, vincristine, dimethylnitrosamine,N-methyl-N′-nitro-Nitrosoguanidine (MNNG), nitrosoguanidine,2-aminopurine, 7, 12 dimethyl-benz(a)anthracene (DMBA), ethylene oxide,hexamethylphosphoramide, bisulfan, diepoxyalkanes (diepoxyoctane (DEO),diepoxybutane (BEB), and the like),2-methoxy-6-chloro-9[3-(ethyl-2-chloro-ethyl)aminopropylamino] acridinedihydrochloride (ICR-170), and formaldehyde. Spontaneous mutations in anSBEII gene that may not have been directly caused by the mutagen canalso be identified.

Any suitable method of plant DNA preparation now known or hereafterdevised may be used to prepare the wheat plant DNA for SBEIIa and SBEIIbmutation screening. For example, see Chen & Ronald, Plant MolecularBiology Reporter 17:53-57, 1999; Stewart and Via, Bio Techniques14:748-749, 1993. Additionally, several commercial kits designed forthis purpose are available, including kits from Qiagen (Valencia,Calif.) and Qbiogene (Carlsbad, Calif.).

In one embodiment, prepared DNA from individual wheat plants are pooledin order to expedite screening for mutations in one or more SBEII genesof the entire population of plants originating from the mutagenizedplant tissue. The size of the pooled group may be dependent upon thesensitivity of the screening method used. Preferably, groups of two ormore individual wheat plants are pooled.

In another embodiment, after the DNA samples are pooled, the pools aresubjected to SBEIIa or SBEIIb sequence-specific amplificationtechniques, such as Polymerase Chain Reaction (PCR). For a generaloverview of PCR, see PCR Protocols: A Guide to Methods and Applications(Innis, Gelfand, Sninsky, and White, eds.), Academic Press, San Diego,1990.

Any primer specific to an SBEIIa locus or an SBEIIb locus or thesequences immediately adjacent to one of these loci may be utilized toamplify the SBEII sequences within the pooled DNA sample. Preferably,the primer is designed to amplify the regions of the SBEII locus whereuseful mutations are most likely to arise. Most preferably, the primeris designed to detect exonic regions of one or more SBEII genes.Additionally, it is preferable for the primer to target knownpolymorphic sites to design genome specific primers in order to easescreening for point mutations in a particular genome. To facilitatedetection of PCR products on a gel, the PCR primer may be labeled usingany conventional or hereafter devised labeling method.

In one embodiment, primers are designed based upon the SBEIIa and SBEIIbhomoeologs (SEQ ID NOs: 1, 3, 5, 7, 9, and 11). Exemplary primers (SEQID NOs: 13-58) that have proven useful in identifying useful mutationswithin the SBEIIa and SBEIIb sequences are shown below in Table 1. Theseprimers are also detailed in the Sequence Listing appended hereto.

TABLE 7 Exemplary Primers SEQ ID NO Region Screened Sequence 13Sbe2a_A_Exon2-3 ACGGCTTTGATCATCTCCTCCCA 14 Sbe2a_A_Exon2-3TTTGTCTCTTTGATGTTCCCCAAAT 15 Sbe2a_A_Exon7-9TATGACCAGAGTATGTCTACAGCTTGGCAAT 16 Sbe2a_A_Exon7-9TGCATCCTAAGTGGGAAACCCTAACCA 17 Sbe2a_A_Exon12-14TCAATTTGGATCAGAGGGGATAGTCCA 18 Sbe2a_A_Exon12-14TGACAAGGTTGCCCATTTCTAATGCAA 19 Sbe2a_B_Exon2-3GATAGCTGGATTAGGCGATCGCCTCAGG 20 Sbe2a_B_Exon2-3TTGGTAGAGGAATTAGCAAAGTAAAATCCA 21 Sbe2a_B_Exon7-9GGTAGAACCTTTTGCATTATGTGTGCTTTTCC 22 Sbe2a_B_Exon7-9GCTACCTCGAAATGCAATGGAAATCTTAGAGAC 23 Sbe2a_B_Exon12-14CCAAGGAGGGAGTGAGGAGCTTGACTT 24 Sbe2a_B_Exon12-14TGTCAGCTTGAATGCCCTTGCACTTCT 25 Sbe2a_D_Exon2-3 GATCGCGCTTCCTGAACCTGTAT26 Sbe2a_D_Exon2-3 CTCAGACCACGAAGGGATCTGTATG 27 Sbe2a_D_Exon7-9ATGAATACGTGCAACACTCCCATCTGC 28 Sbe2a_D_Exon7-9GGAAGCAAAGTTTTGCACTTGCCAATATG 29 Sbe2a_D_Exon10-11CGTCTCCAGCAAGCCATTTCCTACCTTA 30 Sbe2a_D_Exon10-11TTTTGCCACTAGTTTTTGCCAATTTTCC 31 Sbe2a_D_Exon12-14TCAATCAATTTGGATCAGAGGGAACATCA 32 Sbe2a_D_Exon12-14TAGCAGTGCAGGAATTTAAGTTAAACCACTATTACA 33 Sbe2b_A_Exon2-3CTCCCATTCTCGTTTATTCGTAGC 34 Sbe2b_A_Exon2-3 GTTCGGTTACCATGTCACCTCAGAGC35 Sbe2b_A_Exon4-7 GCCAATTGAACAACAATGCCACTTCATT 36 Sbe2b_A_Exon4-7GAGTACCCATTCGCACCTAGATGT 37 Sbe2b_A_Exon7-9 GCCTGTTGCACGAGCCCATTAATTACT38 Sbe2b_A_Exon7-9 TTCGAACAAATGGACACCAGCTTTTGAT 39 Sbe2b_A_Exon10-11TTATATATCAACTTATGAATCCTGAACG 40 Sbe2b_A_Exon10-11GTAAAGTGTTCTTTTAGCAATTTATACAAAC 41 Sbe2b_B_Exon1-3GCCTCCTCATTTCGCTCGCGTGGGTTTAAG 42 Sbe2b_B_Exon1-3AGTGACTATGAACTTCAAGAATTTCGTGATACATCA 43 Sbe2b_B_Exon4-6CTACAAAAAATTGAACAACGATGCCACTTCAT 44 Sbe2b_B_Exon4-6CCAACTATATTTACAGCTCAACTCTGG 45 Sbe2b_B_Exon7-9ACTGATTTTGTTCTTGCAAGACATTCA 46 Sbe2b_B_Exon7-9 CAAATGGACACCAGCTTTTGATGC47 Sbe2b_B_Exon10-11 AAAGTTAGCTATATGCAGTTTAAGTTAATTTACAGGT 48Sbe2b_B_Exon10-11 TGTAAGATGTTCTTTCAGCAATTTATACTA 49 Sbe2b_D_Exon2-3ACGACGCGTGCCGATTCCGTAT 50 Sbe2b_D_Exon2-3GCCATTCACATCTTATCAAAGACTGTAAATTGTTT 51 Sbe2b_D_Exon4-7ATCCTACAAAAAATTGAACAACAATGCCACTTTC 52 Sbe2b_D_Exon4-7ACATGGAGCTACAGTTCAGATGTGC 53 Sbe2b_D_Exon7-9 GCCTGTTGCACGAGCCCATTACTAGAT54 Sbe2b_D_Exon7-9 GGCAATTACTTGTTTCTTTGTGCAATTACTTGTT 55Sbe2b_D_Exon10-11 GTTTTGAATGCTCAAGAGAAGTACTAGT 56 Sbe2b_D_Exon10-11TGTAAGATGTTCTTTCAGCAATTTATACTA 57 Sbe2b_D_Exon12-14TTATGTCTTGGTCCAAAGCCCCTTTTTG 58 Sbe2b_D_Exon12-14TCCACGTCAGGAACTTAGACATGCAACTAT

In another embodiment, the PCR amplification products may be screenedfor SBEII mutations using any method that identifies nucleotidedifferences between wild type and mutant sequences. These may include,for example, without limitation, sequencing, denaturing high pressureliquid chromatography (dHPLC), constant denaturant capillaryelectrophoresis (CDCE), temperature gradient capillary electrophoresis(TGCE) (see Li et al., Electrophoresis 23(10):1499-1511, 2002), or byfragmentation using enzymatic cleavage, such as used in the highthroughput method described by Colbert et al., Plant Physiology126:480-484, 2001. Preferably, the PCR amplification products areincubated with an endonuclease that preferentially cleaves mismatches inheteroduplexes between wild type and mutant sequences.

In another embodiment, cleavage products are electrophoresed using anautomated sequencing gel apparatus, and gel images are analyzed with theaid of a standard commercial image-processing program.

In yet another embodiment, once an M2 plant having a mutated SBEII genesequence is identified, the mutations are analyzed to determine theireffect on the expression, translation, and/or activity of an SBEIIenzyme. In one embodiment, the PCR fragment containing the mutation issequenced, using standard sequencing techniques, in order to determinethe exact location of the mutation in relation to the overall SBEIIsequence. Each mutation is evaluated in order to predict its impact onprotein function (i.e., from completely tolerated to causingloss-of-function) using bioinformatics tools such as SIFT (SortingIntolerant from Tolerant; Ng and Henikoff, Nucleic Acids Research31:3812-3814, 2003), PSSM (Position-Specific Scoring Matrix; Henikoffand Henikoff, Computer Applications in the Biosciences 12:135-143, 1996)and PARSESNP (Taylor and Greene, Nucleic Acids Research 31:3808-3811,2003). For example, a SIFT score that is less than 0.05 and a largechange in PSSM score (e.g., roughly 10 or above) indicate a mutationthat is likely to have a deleterious effect on protein function. Theseprograms are known to be predictive, and it is understood by thoseskilled in the art that the predicted outcomes are not always accurate.

In another embodiment, if the initial assessment of a mutation in the M2plant indicates it to be of a useful nature and in a useful positionwithin an SBEII gene, then further phenotypic analysis of the wheatplant containing that mutation may be pursued. In hexaploid wheat,mutations in each of the A, B and D genomes usually must be combinedbefore a phenotype can be detected. In tetraploid wheat, A and B genomemutations are combined. In addition, the mutation containing plant canbe backcrossed or outcrossed two times or more in order to eliminatebackground mutations at any generation. Then the backcrossed oroutcrossed plant can be self-pollinated or crossed in order to createplants that are homozygous for the SBEII mutations.

Several physical characteristics of these homozygous SBEII mutant plantsare assessed to determine if the mutation results in a useful phenotypicchange in the wheat plant without resulting in undesirable negativeeffects, such as significantly reduced seed yields.

Methods of Producing a Wheat Plant

In another embodiment, the invention relates to a method for producing awheat plant with increased resistant starch levels. In anotherembodiment, the invention relates to a method for producing a wheatplant with an increased proportion of amylose in the starch.

In another embodiment, the invention relates to a method of out-crossingSBEII gene mutations to wild type wheat. In another embodiment, theinvention relates to a method of out-crossing SBEIIa gene mutations towild type wheat. In another embodiment, the invention relates to amethod of out-crossing SBEIIb gene mutations to wild type wheat.

In another embodiment, the invention relates to a method for producing awheat plant having increased amylose content. In still anotherembodiment, the invention relates to a method for producing a wheatplant having reduced activity of one or more SBEII enzymes compared tothe wild type wheat plants.

In one embodiment, the method comprises inducing at least onenon-transgenic mutation in at least one copy of an SBEII gene in plantmaterial or plant parts from a parent wheat plant; growing or using themutagenized plant material to produce progeny wheat plants; analyzingmutagenized plant material and/or progeny wheat plants to detect atleast one mutation in at least one copy of a SBEII gene; and selectingprogeny wheat plants that have at least one mutation in at least onecopy of an SBEII gene.

In another embodiment, the method further comprises crossing progenywheat plants that have at least one mutation in at least one copy of anSBEII gene with other progeny wheat plants that have at least onemutation in a different copy of an SBEII gene. The process ofidentifying progeny wheat plants with mutations and crossing saidprogeny wheat plants with other progeny wheat plants, which havemutations, can be repeated to produce progeny wheat plants with reducedSBEII enzyme activity.

In another embodiment, the level of activity of the SBEII protein in thewheat plant is reduced and selected from the group consisting of 0-2%,2-5%, 5-7%, 7-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%,40-45%, 45-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, 95-99% of thelevel of activity of the SBEII protein in the wild type plant.

In still another embodiment, the level of activity of the SBEIIa proteinin the wheat plant is reduced compared to the wild type plant and isselected from the group consisting of 0-2%, 2-5%, 5-7%, 7-10%, 10-15%,15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, 45-50%, 50-60%, 60-70%,70-80%, 80-90%, 90-95%, 95-99% of the level of activity of the SBEIIaprotein in the wild type plant.

In yet another embodiment, the level of activity of the SBEIIb proteinin the wheat plant is reduced and selected from the group consisting of0-2% 2-5%, 5-7%, 7-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%,40-45%, 45-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, 95-99% of thelevel of activity of the SBEIIb protein in the wild type plant.

A. Methods of Producing a Wheat Plant with One or More Mutations in theSBEIIa Gene in More than One Genome

In still another embodiment, the invention relates to a method forproducing a wheat plant comprising inducing at least one non-transgenicmutation in at least one copy of an SBEIIa gene in plant material from aparent wheat plant that comprises a mutation in an SBEIIa gene; growingor using the mutagenized plant material to produce progeny wheat plants;and selecting progeny wheat plants that have at least one mutation in atleast two copies of an SBEIIa gene.

For example, the parent wheat plant may have a mutation in an SBEIIagene of the A genome. The selected progeny wheat plants may have amutation in an SBEIIa gene of the A genome and one or more mutations inthe SBEIIa gene of the B genome. This example is provided merely forclarification and should not limit the methods disclosed herein.

In yet another embodiment, the invention relates to a method forproducing a wheat plant comprising inducing at least one non-transgenicmutation in at least one copy of an SBEIIa gene in plant material from aparent wheat plant that comprises at least one mutation in two SBEIIagenes; growing or using the mutagenized plant material to produceprogeny wheat plants; and selecting progeny wheat plants that have atleast one mutation in three copies of an SBEIIa gene. In thisembodiment, there would be at least one mutation in the SBEIIa gene ofthe A, B and D genomes.

In another embodiment, the invention relates to a method for producing awheat plant comprising crossing a first wheat plant that has at leastone non-transgenic mutation in a first SBEIIa gene with a second wheatplant that has at least one non-transgenic mutation in a second SBEIIagene; and selecting progeny wheat plants that have at least one mutationin at least two copies of an SBEIIa gene.

In another embodiment, the invention relates to a method for producing awheat plant comprising crossing a first wheat plant that has at leastone non-transgenic mutation in a first and second SBEIIa gene with asecond wheat plant that has at least one non-transgenic mutation in athird SBEIIa gene; and selecting progeny wheat plants that have at leastone mutation in all three copies of an SBEIIa gene. In this embodiment,there would be at least one mutation in the SBEIIa gene of the A, B andD genomes.

In another embodiment, the grain of the wheat plant produced accordingto the methods disclosed herein comprises starch, and the proportion ofamylose in the starch is selected from the group consisting of at least30%, 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, and 60-65% (w/w).In one embodiment, the proportion of amylose in the starch is 47-60%(w/w).

B. Methods of Producing a Wheat Plant with Mutations in the SBEIIb Genein More than One Genome

In still another embodiment, the invention relates to a method forproducing a wheat plant comprising inducing at least one non-transgenicmutation in at least one copy of an SBEIIb gene in plant material from aparent wheat plant that comprises a mutation in an SBEIIb gene; growingor using the mutagenized plant material to produce progeny wheat plants;and selecting progeny wheat plants that have at least one mutation in atleast two copies of an SBEIIb gene.

For example, the parent wheat plant may have a mutation in an SBEIIbgene of the A genome. The selected progeny wheat plants may have amutation in an SBEIIb gene of the A genome and one or more mutations inthe SBEIIb gene of the B genome. This example is provided merely forclarification and should not limit the methods disclosed herein.

In yet another embodiment, the invention relates to a method forproducing a wheat plant comprising inducing at least one non-transgenicmutation in at least one copy of an SBEIIb gene in plant material from aparent wheat plant that comprises at least one mutation in two SBEIIbgenes; growing or using the mutagenized plant material to produceprogeny wheat plants; and selecting progeny wheat plants that have atleast one mutation in three copies of an SBEIIb gene. In thisembodiment, there would be at least one mutation in the SBEIIb gene ofthe A, B and D genomes.

In another embodiment, the invention relates to a method for producing awheat plant comprising crossing a first wheat plant that has at leastone non-transgenic mutation in a first SBEIIb gene with a second wheatplant that has at least one non-transgenic mutation in a second SBEIIbgene; and selecting progeny wheat plants that have at least one mutationin at least two copies of an SBEIIb gene.

In another embodiment, the invention relates to a method for producing awheat plant comprising crossing a first wheat plant that has at leastone non-transgenic mutation in a first and second SBEIIb gene with asecond wheat plant that has at least one non-transgenic mutation in athird SBEIIb gene; and selecting progeny wheat plants that have at leastone mutation in all three copies of an SBEIIb gene. In this embodiment,there would be at least one mutation in the SBEIIb gene of the A, B andD genomes.

In another embodiment, the grain of the wheat plant produced accordingto the methods disclosed herein comprises starch, and the proportion ofamylose in the starch is selected from the group consisting of at least30%, 30-35%, 35-40%, 40-45%, 45-50%, 55-60%, 60-65%, 65-70%, 70-75%,75-80%, 80-85%, 85-90%, 90-95%, and greater than 95% (w/w).

C. Methods of Producing a Wheat Plant with One or More Mutations in theSBEIIa Gene and SBEIIb Gene in More than One Genome

In one embodiment, the invention relates to a method of producing awheat plant with one or more mutations in the SBEIIa gene and one ormore mutations in the SBEIIb gene in one or more than one genome.

In one embodiment, the wheat plant may comprise one mutation in theSBEIIa gene and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 mutationsin the SBEIIb gene. In one embodiment, the wheat plant may comprise 2mutations in the SBEIIa gene and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or morethan 10 mutations in the SBEIIb gene.

In one embodiment, the wheat plant may comprise 3 mutations in theSBEIIa gene and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 mutationsin the SBEIIb gene. In one embodiment, the wheat plant may comprise 4mutations in the SBEIIa gene and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or morethan 10 mutations in the SBEIIb gene. In one embodiment, the wheat plantmay comprise 5 mutations in the SBEIIa gene and 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more than 10 mutations in the SBEIIb gene. In one embodiment,the wheat plant may comprise 6 mutations in the SBEIIa gene and 1, 2, 3,4, 5, 6, 7, 8, 9, 10, or more than 10 mutations in the SBEIIb gene.

In one embodiment, the wheat plant may comprise 7 mutations in theSBEIIa gene and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 mutationsin the SBEIIb gene. In one embodiment, the wheat plant may comprise 8mutations in the SBEIIa gene and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or morethan 10 mutations in the SBEIIb gene. In one embodiment, the wheat plantmay comprise 9 mutations in the SBEIIa gene and 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more than 10 mutations in the SBEIIb gene. In one embodiment,the wheat plant may comprise 10 mutations in the SBEIIa gene and 1, 2,3, 4, 5, 6, 7, 8, 9, 10, or more than 10 mutations in the SBEIIb gene.

In one embodiment, the invention relates to a method for producing awheat plant comprising inducing at least one non-transgenic mutation inat least one copy of an SBEIIa and SBEIIb gene in plant material from aparent wheat plant that comprises a mutation in an SBEIIa and SBEIIbgenes; growing or using the mutagenized plant material to produceprogeny wheat plants; and selecting progeny wheat plants that have atleast one mutation in at least two SBEIIa genes and at least onemutation in at least two SBEIIb genes.

For example, the parent wheat plant may have a mutation in SBEIIa andSBEIIb genes of the A genome. The selected progeny wheat plants may havea mutation in an SBEIIa and SBEIIb gene of the A genome and one or moremutations in the SBEIIa and SBEIIb genes of the B genome. This exampleis provided merely for clarification and should not limit the methodsdisclosed herein.

In yet another embodiment, the invention relates to a method forproducing a wheat plant comprising inducing at least one non-transgenicmutation in at least one copy of SBEIIa and SBEIIb genes in plantmaterial from a parent wheat plant that comprises at least one mutationin two SBEIIa genes and at least one mutation in two SBEIIb genes;growing or using the mutagenized plant material to produce progeny wheatplants; and selecting progeny wheat plants that have at least onemutation in three copies of an SBEIIa gene and at least one mutation inthree copies of an SBEIIb gene. In this embodiment, there would be atleast one mutation in the SBEIIa gene of the A, B and D genomes and atleast one mutation in the SBEIIb gene of the A, B and D genomes.

In another embodiment, the invention relates to a method for producing awheat plant comprising crossing a first wheat plant that has at leastone non-transgenic mutation in a first SBEIIa gene and a first SBEIIbgene with a second wheat plant that has at least one non-transgenicmutation in a second SBEIIa gene and a second SBEIIb gene; and selectingprogeny wheat plants that have at least one mutation in at least twocopies of an SBEIIa and SBEIIb gene.

In another embodiment, the invention relates to a method for producing awheat plant comprising crossing a first wheat plant that has at leastone non-transgenic mutation in a first and second SBEIIa gene and atleast one non-transgenic mutation in a first and second SBEIIb gene witha second wheat plant that has at least one non-transgenic mutation in athird SBEIIa and at least one non-transgenic mutation in a third SBEIIbgene; and selecting progeny wheat plants that have at least one mutationin all three copies of an SBEIIa and SBEIIb gene. In this embodiment,there would be at least one mutation in the SBEIIb gene of the A, B andD genomes.

In another embodiment, the grain of the wheat plant produced accordingto the methods disclosed herein comprises starch, and the proportion ofamylose in the starch is selected from the group consisting of at least30%, 30-35%, 35-40%, 40-45%, 45-50%, and 50-55% (w/w).

Wheat Plant, Wheat Seed and Parts of Wheat Plant

In one embodiment, a wheat plant is produced according to the methodsdisclosed herein. In another embodiment, the wheat plant, wheat seed orparts of a wheat plant have one or more mutations in an SBEII gene. Inanother embodiment, the wheat plant, wheat seed or parts of a wheatplant have one or more mutations in SBEII genes.

In another embodiment, the invention relates to a wheat plant, wheatseed or parts of a wheat plant comprising one or more non-transgenicmutations in the SBEIIa gene. In another embodiment, the inventionrelates to a wheat plant, wheat seed or parts of a wheat plantcomprising at least one non-transgenic mutation in the SBEIIa gene ineach of two genomes. In still another embodiment, the invention relatesto a wheat plant, wheat seed or parts of a wheat plant comprising atleast one non-transgenic mutation in the SBEIIa gene in each of threegenomes.

In one embodiment, the wheat plant, wheat seed or parts of a wheat plantcomprises one or more non-transgenic mutations in both alleles of theSBEIIa gene in the A genome. In another embodiment, the non-transgenicmutations are identical in both alleles of the SBEIIa gene of the Agenome.

In one embodiment, the wheat plant, wheat seed or parts of a wheat plantcomprises one or more non-transgenic mutations in both alleles of theSBEIIa gene in the B genome. In another embodiment, the non-transgenicmutations are identical in both alleles of the SBEIIa gene of the Bgenome.

In one embodiment, the wheat plant, wheat seed or parts of a wheat plantcomprises one or more non-transgenic mutations in both alleles of theSBEIIa gene in the D genome. In another embodiment, the non-transgenicmutations are identical in both alleles of the SBEIIa gene of the Dgenome.

In one embodiment, the invention relates to a wheat plant, wheat seed orparts of a wheat plant comprising a polynucleotide of the SBEIIa gene inthe A genome with one or more non-transgenic mutations listed in Table 1and corresponding to SEQ ID NO: 1. In another embodiment, the wheatplant, wheat seed or parts of the wheat plant comprise a polynucleotidewith one or more non-transgenic mutations listed in Table 1 and is 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orgreater than 99% identical or similar to SEQ ID NO: 1.

In still another embodiment, the wheat plant, wheat seed or parts of awheat plant comprise a polynucleotide with one or more non-transgenicmutations listed in Table 1 that codes for a SBEIIa protein, wherein theSBEIIa protein comprises one or more non-transgenic mutations and is85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or greater than 99% identical or similar to SEQ ID NO: 2.

In one embodiment, the invention relates to a wheat plant, wheat seed orparts of a wheat plant comprising a polynucleotide of the SBEIIa gene inthe B genome with one or more non-transgenic mutations listed in Table 2and corresponding to SEQ ID NO: 3. In another embodiment, the wheatplant, wheat seed or parts of a wheat plant comprises a polynucleotidewith one or more non-transgenic mutations listed in Table 2 is 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orgreater than 99% identical or similar to SEQ ID NO: 3.

In still another embodiment, wheat plant, wheat seed or parts of a wheatplant comprises a polynucleotide with one or more non-transgenicmutations listed in Table 2 and codes for a SBEIIa protein, wherein theSBEIIa protein comprises one or more non-transgenic mutations and is85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or greater than 99% identical or similar to SEQ ID NO: 4.

In one embodiment, the invention relates to a wheat plant, wheat seed orparts of a wheat plant comprising a polynucleotide of the SBEIIa gene ofthe D genome with one or more non-transgenic mutations listed in Table 3and corresponding to SEQ ID NO: 5. In another embodiment, the wheatplant, wheat seed or parts of a wheat plant comprise a polynucleotidewith one or more non-transgenic mutations listed in Table 3 and is 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orgreater than 99% identical or similar to SEQ ID NO: 5.

In still another embodiment, the wheat plant, wheat seed or parts of awheat plant comprises a polynucleotide with one or more non-transgenicmutations listed in Table 3 and codes for a SBEIIa protein, wherein theSBEIIa protein comprises one or more non-transgenic mutations and is85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or greater than 99% identical or similar to SEQ ID NO: 6.

In still another embodiment, the invention relates to a wheat plant,wheat seed or parts of a wheat plant comprising one or morenon-transgenic mutations in the SBEIIb gene. In another embodiment, theinvention relates to a wheat plant, wheat seed or parts of a wheat plantcomprising at least one non-transgenic mutation in the SBEIIb gene ineach of two genomes. In still another embodiment, the invention relatesto a wheat plant, wheat seed or parts of a wheat plant comprising atleast one non-transgenic mutation in the SBEIIb gene in each of threegenomes.

In one embodiment, the wheat plant, wheat seed or parts of a wheat plantcomprises one or more non-transgenic mutations in both alleles of theSBEIIb gene. In one embodiment, the wheat plant, wheat seed or parts ofa wheat plant comprises one or more non-transgenic mutations in bothalleles of the SBEIIb gene of the A genome. In another embodiment, thenon-transgenic mutations are identical in both alleles of the SBEIIbgene of the A genome.

In one embodiment, the wheat plant, wheat seed or parts of a wheat plantcomprises one or more non-transgenic mutations in both alleles of theSBEIIb gene of the B genome. In another embodiment, the non-transgenicmutations are identical in both alleles of the SBEIIb gene of the Bgenome.

In one embodiment, the wheat plant, wheat seed or parts of a wheat plantcomprises one or more non-transgenic mutations in both alleles of theSBEIIb gene of the D genome. In another embodiment, the non-transgenicmutations are identical in both alleles of the SBEIIb gene of the Dgenome.

In one embodiment, the invention relates to a wheat plant, wheat seed orparts of a wheat plant comprising a polynucleotide of the SBEIIb gene ofthe A genome with one or more non-transgenic mutations listed in Table 4and corresponding to SEQ ID NO: 7. In another embodiment, the wheatplant, wheat seed or parts of a wheat plant comprises a polynucleotidewith one or more non-transgenic mutations listed in Table 4 and is 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orgreater than 99% identical or similar to SEQ ID NO: 7.

In still another embodiment, the wheat plant, wheat seed or parts of awheat plant comprise a polynucleotide with one or more non-transgenicmutations listed in Table 4 that codes for a SBEIIb protein, wherein theSBEIIb protein comprises one or more non-transgenic mutations and is85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or greater than 99% identical or similar to SEQ ID NO: 8.

In one embodiment, the invention relates to a wheat plant, wheat seed orparts of a wheat plant comprising a polynucleotide of the SBEIIb gene ofthe B genome with one or more non-transgenic mutations listed in Table 5and corresponding to SEQ ID NO: 9. In another embodiment, the wheatplant, wheat seed or parts of a wheat plant comprise a polynucleotidewith one or more non-transgenic mutations listed in Table 5 and is 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orgreater than 99% identical or similar to SEQ ID NO: 9.

In still another embodiment, the wheat plant, wheat seed or parts of awheat plant comprise a polynucleotide with one or more non-transgenicmutations listed in Table 5 that codes for a SBEIIb protein, wherein theSBEIIb protein comprises one or more non-transgenic mutations and is85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or greater than 99% identical or similar to SEQ ID NO: 10.

In one embodiment, the invention relates to wheat plant, wheat seed orparts of a wheat plant comprising a polynucleotide of the SBEIIb gene ofthe D genome with one or more non-transgenic mutations listed in Table 6and corresponding to SEQ ID NO: 11. In another embodiment, the wheatplant, wheat seed or parts of a wheat plant comprise a polynucleotidewith one or more non-transgenic mutations listed in Table 6 and is 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orgreater than 99% identical or similar to SEQ ID NO: 11.

In still another embodiment, the wheat plant, wheat seed or parts of awheat plant comprise a polynucleotide with one or more non-transgenicmutations listed in Table 6 that codes for a SBEIIb protein, wherein theSBEIIb protein comprises one or more non-transgenic mutations and is85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or greater than 99% identical or similar to SEQ ID NO: 12.

In another embodiment, the invention relates to a wheat plant, wheatseed or parts of a wheat plant comprising one or more non-transgenicmutations in the SBEIIa and SBEIIb genes. In another embodiment, theinvention relates to a wheat plant, wheat seed or parts of a wheat plantcomprising at least one non-transgenic mutation in the SBEIIa and SBEIIbgenes in each of two genomes. In still another embodiment, the inventionrelates to a wheat plant, wheat seed or parts of a wheat plantcomprising at least one non-transgenic mutation in the SBEIIa and SBEIIbgenes in each of three genomes.

In still another embodiment, the invention relates to a wheat plant,wheat seed or parts of a wheat plant comprising at least onenon-transgenic mutation in the SBEIIa gene in each of three genomes andone or more non-transgenic mutation in the SBEIIb gene.

In another embodiment, the wheat plant, wheat seed or parts of a wheatplant has one or more mutations in the SBEII gene including but notlimited to one or more mutations enumerated in Tables 1-6 and 8-12 andcorresponding mutations in the homoeologues. A wheat plant, wheat seedor parts of a wheat plant can be generated having 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 orgreater than 25 of the mutations disclosed herein including but notlimited to the mutations disclosed in Tables 1-6 and 8-12, as well asmutations in the corresponding homoeologues.

In another embodiment, a wheat plant, wheat seed or parts of a wheatplant comprising one or more non-transgenic mutations in an SBEII gene,including but not limited to the mutation listed in Tables 1-6 and 8-12and the mutations in the corresponding homoeologues, has an increasedproportion of amylose in starch as compared to the same wheat cultivarwithout the mutations in the SBEII gene. In yet another embodiment, theproportion of amylose in the starch is selected from the groupconsisting of at least 10-15%, 16-20%, 21-25%, 26-30%, 31-35%, 36-40%,41-45%, 46-50%, 51-55%, 56-60%, 61-65%, 66-70%, 71-75%, 76-80%, 81-85%,86-90%, 91-95%, 96%, 97%, 98%, 99%, and greater than 99% (w/w).

Grain, Flour and Starch

In another embodiment, the invention relates to a wheat grain, flour orstarch comprising one or more non-transgenic mutations in the SBEIIgene. In another embodiment, the invention relates to wheat graincomprising an embryo, wherein the embryo comprises one or morenon-transgenic mutations in an SBEII gene.

In another embodiment, the wheat grain, flour or starch comprises one ormore non-transgenic mutations in the SBEIIa and/or the SBEIIb genesincluding but not limited to the mutations recited in Tables 1-6 and8-12 and the corresponding mutations in homoeologues.

In still another embodiment, the invention relates to a wheat grain,flour or starch comprising one or more non-transgenic mutations in theSBEIIa gene. In another embodiment, the invention relates to a wheatgrain or flour comprising at least one non-transgenic mutation in theSBEIIa gene in each of two genomes. In still another embodiment, theinvention relates to a wheat grain or flour comprising at least onenon-transgenic mutation in the SBEIIa gene in each of three genomes.

In one embodiment, the wheat grain, flour or starch comprises one ormore non-transgenic mutations in both alleles of the SBEIIa gene in theA genome. In another embodiment, the non-transgenic mutations areidentical in both alleles of the SBEIIa gene of the A genome.

In one embodiment, the wheat grain, flour or starch comprises one ormore non-transgenic mutations in both alleles of the SBEIIa gene in theB genome. In another embodiment, the non-transgenic mutations areidentical in both alleles of the SBEIIa gene of the B genome.

In one embodiment, the wheat grain, flour or starch comprises one ormore non-transgenic mutations in both alleles of the SBEIIa gene in theD genome. In another embodiment, the non-transgenic mutations areidentical in both alleles of the SBEIIa gene of the D genome.

In one embodiment, the invention relates to wheat grain, wheat flour orstarch comprising a polynucleotide of the SBEIIa gene in the A genomewith one or more non-transgenic mutations listed in Table 1 andcorresponding to SEQ ID NO: 1. In another embodiment, the wheat grain orwheat flour comprise a polynucleotide with one or more non-transgenicmutations listed in Table 1 and is 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than 99% identical orsimilar to SEQ ID NO: 1.

In still another embodiment, wheat grain, wheat flour or starch comprisea polynucleotide with one or more non-transgenic mutations listed inTable 1 that codes for a SBEIIa protein, wherein the SBEIIa proteincomprises one or more non-transgenic mutations and is 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greaterthan 99% identical or similar to SEQ ID NO: 2.

In one embodiment, the invention relates to wheat grain, wheat flour orstarch comprising a polynucleotide of the SBEIIa gene in the B genomewith one or more non-transgenic mutations listed in Table 2 andcorresponding to SEQ ID NO: 3. In another embodiment, the wheat grain orwheat flour comprises a polynucleotide with one or more non-transgenicmutations listed in Table 2 is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than 99% identical orsimilar to SEQ ID NO: 3.

In still another embodiment, wheat grain, wheat flour or starch comprisea polynucleotide with one or more non-transgenic mutations listed inTable 2 and codes for a SBEIIa protein, wherein the SBEIIa proteincomprises one or more non-transgenic mutations and is 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greaterthan 99% identical or similar to SEQ ID NO: 4.

In one embodiment, the invention relates to wheat grain, wheat flour orstarch comprising a polynucleotide of the SBEIIa gene of the D genomewith one or more non-transgenic mutations listed in Table 3 andcorresponding to SEQ ID NO: 5. In another embodiment, the wheat grain orwheat flour comprise a polynucleotide with one or more non-transgenicmutations listed in Table 3 and is 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than 99% identical orsimilar to SEQ ID NO: 5.

In still another embodiment, wheat grain, wheat flour or starch comprisea polynucleotide with one or more non-transgenic mutations listed inTable 3 and codes for a SBEIIa protein, wherein the SBEIIa proteincomprises one or more non-transgenic mutations and is 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greaterthan 99% identical or similar to SEQ ID NO: 6.

In still another embodiment, the invention relates to a wheat grain,flour or starch comprising one or more non-transgenic mutations in theSBEIIb gene. In another embodiment, the invention relates to a wheatplant comprising at least one non-transgenic mutation in the SBEIIb genein each of two genomes. In still another embodiment, the inventionrelates to a wheat plant comprising at least one non-transgenic mutationin the SBEIIb gene in each of three genomes.

In one embodiment, the wheat grain, flour or starch comprises one ormore non-transgenic mutations in both alleles of the SBEIIb gene. In oneembodiment, the wheat grain, flour or starch comprises one or morenon-transgenic mutations in both alleles of the SBEIIb gene in each oftwo genomes. In one embodiment, the wheat grain, flour or starchcomprises one or more non-transgenic mutations in both alleles of theSBEIIb gene in each of three genomes.

In one embodiment, the wheat grain, flour or starch comprises one ormore non-transgenic mutations in both alleles of the SBEIIb gene. In oneembodiment, the wheat grain, flour or starch comprises one or morenon-transgenic mutations in both alleles of the SBEIIb gene of the Agenome. In another embodiment, the non-transgenic mutations areidentical in both alleles of the SBEIIb gene of the A genome.

In one embodiment, the wheat grain, flour or starch comprises one ormore non-transgenic mutations in both alleles of the SBEIIb gene of theB genome. In another embodiment, the non-transgenic mutations areidentical in both alleles of the SBEIIb gene of the B genome.

In one embodiment, the wheat grain, flour or starch comprises one ormore non-transgenic mutations in both alleles of the SBEIIb gene of theD genome. In another embodiment, the non-transgenic mutations areidentical in both alleles of the SBEIIb gene of the D genome.

In one embodiment, the invention relates to a wheat grain, wheat flouror starch comprising a polynucleotide of the SBEIIb gene of the A genomewith one or more non-transgenic mutations listed in Table 4 andcorresponding to SEQ ID NO: 7. In another embodiment, the wheat grain,wheat flour or starch comprises a polynucleotide with one or morenon-transgenic mutations listed in Table 4 and is 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than99% identical or similar to SEQ ID NO: 7.

In still another embodiment, the wheat grain, wheat flour or starchcomprise a polynucleotide with one or more non-transgenic mutationslisted in Table 4 that codes for a SBEIIb protein, wherein the SBEIIbprotein comprises one or more non-transgenic mutations and is 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orgreater than 99% identical or similar to SEQ ID NO: 8.

In one embodiment, the invention relates to wheat grain, wheat flour orstarch comprising a polynucleotide of the SBEIIb gene of the B genomewith one or more non-transgenic mutations listed in Table 5 andcorresponding to SEQ ID NO: 9. In another embodiment, the wheat grain,wheat flour or starch comprise a polynucleotide with one or morenon-transgenic mutations listed in Table 5 and is 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than99% identical or similar to SEQ ID NO: 9.

In still another embodiment, the wheat grain, wheat flour or starchcomprise a polynucleotide with one or more non-transgenic mutationslisted in Table 5 that codes for a SBEIIb protein, wherein the SBEIIbprotein comprises one or more non-transgenic mutations and is 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orgreater than 99% identical or similar to SEQ ID NO: 10.

In one embodiment, the invention relates to wheat grain, wheat flour orstarch comprising a polynucleotide of the SBEIIb gene of the D genomewith one or more non-transgenic mutations listed in Table 6 andcorresponding to SEQ ID NO: 11. In another embodiment, the wheat grain,wheat flour or starch comprise a polynucleotide with one or morenon-transgenic mutations listed in Table 6 and is 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than99% identical or similar to SEQ ID NO: 11.

In still another embodiment, the wheat grain, wheat flour or starchcomprise a polynucleotide with one or more non-transgenic mutationslisted in Table 6 that codes for a SBEIIb protein, wherein the SBEIIbprotein comprises one or more non-transgenic mutations and is 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orgreater than 99% identical or similar to SEQ ID NO: 12.

In another embodiment, the invention relates to a wheat grain, flour orstarch comprising one or more non-transgenic mutations in the SBEIIagene and one or more non-transgenic mutations in the SBEIIb genes. Inanother embodiment, the invention relates to a wheat grain, flour orstarch comprising at least one non-transgenic mutation in the SBEIIa andSBEIIb genes in each of two genomes. In still another embodiment, theinvention relates to a wheat grain, flour or starch comprising at leastone non-transgenic mutation in the SBEIIa and SBEIIb genes in each ofthree genomes.

In still another embodiment, the invention relates to a wheat grain,flour or starch comprising at least one non-transgenic mutation in theSBEIIa gene in each of three genomes and one or more non-transgenicmutation in the SBEIIb gene.

In one embodiment, the wheat grain, flour or starch comprises one ormore non-transgenic mutations in both alleles of the SBEIIa gene and theSBEIIb gene of the A genome. In another embodiment, the non-transgenicmutations are identical in both alleles of the SBEIIa gene and theSBEIIb gene of the A genome.

In one embodiment, the wheat grain, flour or starch comprises one ormore non-transgenic mutations in both alleles of the SBEIIa gene and theSBEIIb gene of the B genome. In another embodiment, the non-transgenicmutations are identical in both alleles of the SBEIIa gene and theSBEIIb gene of the B genome.

In one embodiment, the wheat grain, flour or starch comprises one ormore non-transgenic mutations in both alleles of the SBEIIa gene and theSBEIIb gene of the D genome. In another embodiment, the non-transgenicmutations are identical in both alleles of the SBEIIa gene and theSBEIIb gene of the D genome.

In still another embodiment, the invention relates to wheat grain orflour comprising an endosperm and a reduced gene expression level,activity or expression level and activity of the SBEII gene as comparedto wild type wheat grain or flour.

In still another embodiment, the invention relates to wheat grain orflour comprising an endosperm and a reduced expression level, activityor expression level and activity of the SBEII protein as compared towild type wheat grain or flour. In still another embodiment, theinvention relates to wheat grain or flour comprising an endosperm and areduced expression level, activity or expression level and activity ofthe SBEIIa protein as compared to wild type wheat grain or flour. In yetanother embodiment, the invention relates to wheat grain or flourcomprising an endosperm and a reduced expression level, activity orexpression level and activity of the SBEIIb protein as compared to wildtype wheat grain or flour.

In yet another embodiment, the invention relates to wheat grain or flourcomprising an altered starch component as compared to starch from wildtype wheat grain or flour. In another embodiment, the wheat grain orflour comprises starch with a percentage of amylose selected from thegroup consisting of: 25-30%, 30-35%, 35-40%, 45-50%, 50-55%, 55-60%,60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, and greater than95% as compared to wild type grain or flour.

Food Products

In one embodiment, the invention is directed to a flour or other productproduced from the grain or flour discussed above. In anotherembodiments, the flour, the coarse fraction or purified starch may be acomponent of a food product.

The food product includes but is not limited to a bagel, a biscuit, abread, a bun, a croissant, a dumpling, an English muffin, a muffin, apita bread, a quickbread, a refrigerated/frozen dough products, dough,baked beans, a burrito, chili, a taco, a tamale, a tortilla, a pot pie,a ready to eat cereal, a ready to eat meal, stuffing, a microwaveablemeal, a brownie, a cake, a cheesecake, a coffee cake, a cookie, adessert, a pastry, a sweet roll, a candy bar, a pie crust, pie filling,baby food, a baking mix, a batter, a breading, a gravy mix, a meatextender, a meat substitute, a seasoning mix, a soup mix, a gravy, aroux, a salad dressing, a soup, sour cream, a noodle, a pasta, ramennoodles, chow mein noodles, lo mein noodles, an ice cream inclusion, anice cream bar, an ice cream cone, an ice cream sandwich, a cracker, acrouton, a doughnut, an egg roll, an extruded snack, a fruit and grainbar, a microwaveable snack product, a nutritional bar, a pancake, apar-baked bakery product, a pretzel, a pudding, a granola-based product,a snack chip, a snack food, a snack mix, a waffle, a pizza crust, animalfood or pet food.

In one embodiment, the flour is a whole grain flour (ex.—anultrafine-milled whole grain flour, such as an ultrafine-milled wholegrain wheat flour). In one embodiment, the whole grain flour includes arefined flour constituent (ex.—refined wheat flour or refined flour) anda coarse fraction (ex.—an ultrafine-milled coarse fraction). Refinedwheat flour may be flour which is prepared, for example, by grinding andbolting (sifting) cleaned wheat. The Food and Drug Administration (FDA)requires flour to meet certain particle size standards in order to beincluded in the category of refined wheat flour. The particle size ofrefined wheat flour is described as flour in which not less than 98%passes through a cloth having openings not larger than those of wovenwire cloth designated “212 micrometers (U.S. Wire 70).”

In another embodiment, the coarse fraction includes at least one of:bran and germ. For instance, the germ is an embryonic plant found withinthe wheat kernel. The germ includes lipids, fiber, vitamins, protein,minerals and phytonutrients, such as flavonoids. The bran may includeseveral cell layers and has a significant amount of lipids, fiber,vitamins, protein, minerals and phytonutrients, such as flavonoids.

For example, the coarse fraction or whole grain flour or refined flourof the present invention may be used in various amounts to replacerefined or whole grain flour in baked goods, snack products, and foodproducts. The whole grain flour (i.e.—ultrafine-milled whole grainflour) may also be marketed directly to consumers for use in theirhomemade baked products. In an exemplary embodiment, a granulationprofile of the whole grain flour is such that 98% of particles by weightof the whole grain flour are less than 212 micrometers.

In another embodiment, the whole grain flour or coarse fraction orrefined flour may be a component of a nutritional supplement. Thenutritional supplement may be a product that is added to the dietcontaining one or more ingredients, typically including: vitamins,minerals, herbs, amino acids, enzymes, antioxidants, herbs, spices,probiotics, extracts, prebiotics and fiber.

In a further embodiment, the nutritional supplement may include anyknown nutritional ingredients that will aid in the overall health of anindividual, examples include but are not limited to vitamins, minerals,other fiber components, fatty acids, antioxidants, amino acids,peptides, proteins, lutein, ribose, omega-3 fatty acids, and/or othernutritional ingredients. Because of the high nutritional content of theendosperm of the present invention, there may be many uses that confernumerous benefits to an individual, including, delivery of fiber andother essential nutrients, increased digestive function and health,weight management, blood sugar management, heart health, diabetes riskreduction, potential arthritis risk reduction, and overall health andwellness for an individual.

In still another embodiments, the whole grain flour or coarse fractionor refined flour may be a component of a dietary supplement. The Code ofFederal Regulations defines a dietary supplement as a product that isintended to supplement the diet and contains one or more dietaryingredients including: vitamins, minerals, herbs, botanicals, aminoacids, and other substances or their constituents; is intended to betaken by mouth as a pill, capsule, tablet, or liquid; and is labeled onthe front panel as being a dietary supplement.

In yet another embodiment, the whole grain flour or coarse fraction orrefined flour may be a fiber supplement or a component thereof. Thefiber supplement may be delivered in, but is not limited to thefollowing forms: instant beverage mixes, ready-to-drink beverages,nutritional bars, wafers, cookies, crackers, gel shots, capsules, chews,chewable tablets, and pills. One embodiment delivers the fibersupplement in the form of a flavored shake or malt type beverage.

In another embodiment, the whole grain flour or coarse fraction orrefined flour may be included as a component of a digestive supplement.The whole grain flour or coarse fraction or refined flour may be acomponent of a digestive supplement alone or in combination with one ormore prebiotic compounds and/or probiotic organisms. Prebiotic compoundsare non-digestible food ingredients that may beneficially affect thehost by selectively stimulating the growth and/or the activity of alimited number of microorganisms in the colon. Examples of prebioticcompounds within the scope of the invention, may include, but are notlimited to: oligosaccharides and inulins.

Probiotics are microorganisms which, when administered in adequateamounts, confer a health benefit on the host. Probiotic organismsinclude, but are not limited to: Lactobacillus, Bifidobacteria,Escherichia, Clostridium, Lactococcus, Streptococcus, Enterococcus, andSaccharomyces.

In yet another embodiment, the whole grain flour or coarse fraction orrefined flour may be included as a component of a functional food. TheInstitute of Food Technologists defines functional foods as, foods andfood components that provide a health benefit beyond basic nutrition.This includes conventional foods, fortified, enriched, or enhancedfoods, and dietary supplements. The whole grain flour and coarsefraction or refined flour include numerous vitamins and minerals, havehigh oxygen radical absorption capacities, and are high in fiber, makingthem ideally suited for use in/as a functional food.

In another embodiment, the whole grain flour or coarse fraction orrefined flour may be used in medical foods. Medical food is defined as afood that is formulated to be consumed or administered entirely underthe supervision of a physician and which is intended for the specificdietary management of a disease or condition for which distinctivenutritional requirements, based on recognized scientific principles, areestablished by medical evaluation. The nutrient contents and antioxidantcapacities of the whole grain flour and coarse fraction or refined flourmake them ideal for use in medical foods.

In yet another embodiment, the whole grain flour or coarse fraction orrefined flour may also be used in pharmaceuticals. The whole grain flourand coarse fraction or refined flour are high in fiber and have a veryfine granulation making them suitable for use as a carrier inpharmaceuticals.

In still another embodiment, delivery of the whole grain flour or coarsefraction or refined flour as a nutritional supplement, dietarysupplement or digestive supplement is contemplated via deliverymechanisms where the whole grain flour or coarse fraction is the singleingredient or one of many nutritional ingredients. Examples of deliverymechanisms include but are not limited to: instant beverage mixes,ready-to-drink beverages, nutritional bars, wafers, cookies, crackers,gel shots, capsules, and chews.

In yet another embodiment, a milling process may be used to make amulti-wheat flour, or a multi-grain coarse fraction. In one embodiment,bran and germ from one type of wheat may be ground and blended withground endosperm or whole grain wheat flour of another type of wheat.Alternatively bran and germ of one type of grain may be ground andblended with ground endosperm or whole grain flour of another type ofgrain.

In still another embodiment, bran and germ from a first type of wheat orgrain may be blended with bran and germ from a second type of wheat orgrain to produce a multi-grain coarse fraction. It is contemplated thatthe invention encompasses mixing any combination of one or more of bran,germ, endosperm, and whole grain flour of one or more grains. Thismulti-grain, multi-wheat approach may be used to make custom flour andcapitalize on the qualities and nutritional contents of multiple typesof grains or wheats to make one flour.

The whole grain flour of the invention may be produced via a variety ofmilling processes. One exemplary process involves grinding grain in asingle stream without separating endosperm, bran, and germ of the graininto separate streams. Clean and tempered grain is conveyed to a firstpassage grinder, such as a hammermill, roller mill, pin mill, impactmill, disc mill, air attrition mill, gap mill, or the like.

After grinding, the grain is discharged and conveyed to a sifter. Anysifter known in the art for sifting a ground particle may be used.Material passing through the screen of the sifter is the whole grainflour of the invention and requires no further processing. Material thatremains on the screen is referred to as a second fraction. The secondfraction requires additional particle reduction. Thus, this secondfraction may be conveyed to a second passage grinder.

After grinding, the second fraction may be conveyed to a second sifter.Material passing through the screen of the second sifter is the wholegrain flour. The material that remains on the screen is referred to asthe fourth fraction and requires further processing to reduce theparticle size. The fourth fraction on the screen of the second sifter isconveyed back into either the first passage grinder or the secondpassage grinder for further processing via a feedback loop.

It is contemplated that the whole grain flour, coarse fraction, purifiedstarch and/or grain products of the invention may be produced by anumber of milling processes known in the art.

Plant Breeding

In another embodiment, this invention is directed to methods for plantbreeding using wheat plants and plant parts with one or morenon-transgenic mutations in the SBEII gene.

One such embodiment is the method of crossing wheat variety with one ormore non-transgenic mutations in the SBEII gene with another variety ofwheat to form a first generation population of F1 plants. The populationof first generation F1 plants produced by this method is also anembodiment of the invention. This first generation population of F1plants will comprise an essentially complete set of the alleles of wheatvariety with one or more non-transgenic mutations in the SBEII gene. Oneof ordinary skill in the art can utilize either breeder books ormolecular methods to identify a particular F1 plant produced using wheatvariety with one or more non-transgenic mutations in the SBEII gene, andany such individual plant is also encompassed by this invention. Theseembodiments also cover use of transgenic or backcross conversions ofwheat varieties with one or more mutations in the SBEII gene to producefirst generation F1 plants.

In another embodiment, the invention relates to a method of developing aprogeny wheat plant. A method of developing a progeny wheat plantcomprises crossing a wheat variety with one or more non-transgenicmutations in the SBEII gene with a second wheat plant and performing abreeding method. A specific method for producing a line derived fromwheat variety with one or more non-transgenic mutations in the SBEIIgene is as follows.

One of ordinary skill in the art would cross wheat variety with one ormore non-transgenic mutations in the SBEII gene with another variety ofwheat, such as an elite variety. The F1 seed derived from this crosswould be grown to form a homogeneous population. The F1 seed wouldcontain one set of the alleles from wheat variety with one or morenon-transgenic mutations in the SBEII gene and one set of the allelesfrom the other wheat variety.

The F1 genome would be made-up of 50% wheat variety with one or morenon-transgenic mutations in the SBEII gene and 50% of the other elitevariety. The F1 seed would be grown to form F2 seed. The F1 seed couldbe allowed to self, or bred with another wheat cultivar.

On average the F2 seed would have derived 50% of its alleles from wheatvariety with one or more non-transgenic mutations in the SBEII gene and50% from the other wheat variety, but various individual plants from thepopulation would have a much greater percentage of their alleles derivedfrom wheat variety with one or more non-transgenic mutations in theSBEII gene (Wang J. and R. Bernardo, 2000, Crop Sci. 40:659-665 andBernardo, R. and A. L. Kahler, 2001, Theor. Appl. Genet. 102:986-992).

The F2 seed would be grown and selection of plants would be made basedon visual observation and/or measurement of traits and/or markerassisted selection. The wheat variety with one or more non-transgenicmutations in the SBEII gene-derived progeny that exhibit one or more ofthe desired wheat variety with one or more non-transgenic mutations inthe SBEII gene-derived traits would be selected and each plant would beharvested separately. This F3 seed from each plant would be grown inindividual rows and allowed to self. Then selected rows or plants fromthe rows would be harvested and threshed individually. The selectionswould again be based on visual observation and/or measurements fordesirable traits of the plants, such as one or more of the desirablewheat variety with one or more non-transgenic mutations in the SBEIIgene-derived traits.

The process of growing and selection would be repeated any number oftimes until a homozygous wheat variety with one or more non-transgenicmutations in the SBEII gene-derived wheat plant is obtained. Thehomozygous wheat variety with one or more non-transgenic mutations inthe SBEII gene-derived wheat plant would contain desirable traitsderived from wheat variety with one or more non-transgenic mutations inthe SBEII gene, some of which may not have been expressed by the otheroriginal wheat variety to which wheat variety with one or morenon-transgenic mutations in the SBEII gene was crossed and some of whichmay have been expressed by both wheat varieties but now would be at alevel equal to or greater than the level expressed in wheat variety withone or more non-transgenic mutations in the SBEII gene.

The breeding process, of crossing, selfing, and selection may berepeated to produce another population of wheat variety with one or morenon-transgenic mutations in the SBEII gene-derived wheat plants with, onaverage, 25% of their genes derived from wheat variety with one or morenon-transgenic mutations in the SBEII gene, but various individualplants from the population would have a much greater percentage of theiralleles derived from wheat variety with one or more non-transgenicmutations in the SBEII gene. Another embodiment of the invention is ahomozygous wheat variety with one or more non-transgenic mutations inthe SBEII gene-derived wheat plant that has received wheat variety withone or more non-transgenic mutations in the SBEII gene-derived traits.

The invention is further described by the following paragraphs.

1. A polynucleotide encoding an SBEIIa polypeptide comprising atryptophan to a stop mutation at an amino acid corresponding to aminoacid position 436 of SEQ ID NO: 2.

2. The polynucleotide of paragraph 1, wherein the SBEIIa polypeptidefurther comprises an amino acid sequence having at least 95% identity orsimilarity to SEQ ID NO: 2.

3. The polynucleotide of any of paragraphs 1-2, wherein the SBEIIapolypeptide further comprises an amino acid sequence having at least 97%identity or similarity to SEQ ID NO: 2.

4. The polynucleotide of any of paragraphs 1-3, wherein the SBEIIapolypeptide further comprises an amino acid sequence having at least 99%identity or similarity to SEQ ID NO: 2.

5. The polynucleotide of any of paragraphs 1-4 comprising a guanine toadenine mutation at a nucleotide position corresponding to nucleotideposition 5267 of SEQ ID NO: 1.

6. The polynucleotide of any of paragraphs 1-5 further comprising atleast 95% identity or similarity to SEQ ID NO: 1.

7. The polynucleotide of any of paragraphs 1-6 further comprising atleast 97% identity or similarity to SEQ ID NO: 1.

8. The polynucleotide of any of paragraphs 1-7 further comprising atleast 99% identity or similarity to SEQ ID NO: 1.

9. A polypeptide comprising an amino acid sequence having at least 95%identity or similarity to SEQ ID NO:2, wherein the polypeptide furthercomprises a tryptophan to a stop mutation at amino acid position 436 ofSEQ ID NO: 2.

10. The polypeptide of paragraph 9 further comprising an amino acidsequence having at least 97% sequence identity or similarity to SEQ IDNO:2.

11. The polypeptide of any of paragraphs 9-10 further comprising anamino acid sequence having at least 99% sequence identity or similarityto SEQ ID NO:2.

12. The polypeptide of any of paragraphs 9-11 further comprising anamino acid sequence of SEQ ID NO:2 with a tryptophan to a stop mutationat amino acid position 436 or a fragment thereof having starch branchingenzyme activity.

13. The polypeptide of any of paragraphs 1-12 further comprising anamino acid sequence of SEQ ID NO:2 with a tryptophan to a stop mutationat amino acid position 436.

14. A polynucleotide encoding an SBEIIa polypeptide comprising atryptophan to a stop mutation at an amino acid corresponding to aminoacid position 436 of SEQ ID NO: 4.

15. The polynucleotide of paragraph 14, wherein the SBEIIa polypeptidefurther comprises an amino acid sequence having at least 95% identity orsimilarity to SEQ ID NO: 4.

16. The polynucleotide of any of paragraphs 14-15, wherein the SBEIIapolypeptide further comprises an amino acid sequence having at least 97%identity or similarity to SEQ ID NO: 4.

17. The polynucleotide of any of paragraphs 14-16, wherein the SBEIIapolypeptide further comprises an amino acid sequence having at least 99%identity or similarity to SEQ ID NO: 4.

18. The polynucleotide of any of paragraphs 14-17 comprising a guanineto adenine mutation at a nucleotide position corresponding to nucleotideposition 5038 of SEQ ID NO: 3.

19. The polynucleotide of any of paragraphs 14-18 further comprising atleast 95% identity or similarity to SEQ ID NO: 3.

20. The polynucleotide of any of paragraphs 14-19 further comprising atleast 97% identity or similarity to SEQ ID NO: 3.

21. The polynucleotide of any of paragraphs 14-20 further comprising atleast 99% identity or similarity to SEQ ID NO: 3.

22. A polypeptide comprising an amino acid sequence having at least 95%identity or similarity to SEQ ID NO:4, wherein the polypeptide furthercomprises a tryptophan to a stop mutation at amino acid position 436 ofSEQ ID NO: 4.

23. The polypeptide of paragraph 22 further comprising an amino acidsequence having at least 97% sequence identity or similarity to SEQ IDNO:4.

24. The polypeptide of any of paragraphs 22-23 further comprising anamino acid sequence having at least 99% sequence identity or similarityto SEQ ID NO:4.

25. The polypeptide of any of paragraphs 22-24 comprising an amino acidsequence of SEQ ID NO:4 with a tryptophan to a stop mutation at aminoacid position 436 or a fragment thereof having starch branching enzymeactivity.

26. The polypeptide of any of paragraphs 22-25 comprising an amino acidsequence of SEQ ID NO:4 with a tryptophan to a stop mutation at aminoacid position 436.

27. A polynucleotide encoding an SBEIIa polypeptide comprising atryptophan to a stop mutation at an amino acid corresponding to aminoacid position 432 of SEQ ID NO: 6.

28. The polynucleotide of paragraph 27, wherein the SBEIIa polypeptidefurther comprises an amino acid sequence having at least 95% identity orsimilarity to SEQ ID NO: 6.

29. The polynucleotide of any of paragraphs 27-28, wherein the SBEIIapolypeptide further comprises an amino acid sequence having at least 97%identity or similarity to SEQ ID NO: 6.

30. The polynucleotide of any of paragraphs 27-29, wherein the SBEIIapolypeptide further comprises an amino acid sequence having at least 99%identity or similarity to SEQ ID NO: 6.

31. The polynucleotide of any of paragraphs 27-30 comprising a guanineto adenine mutation at a nucleotide position corresponding to nucleotideposition 6305 of SEQ ID NO: 5.

32. The polynucleotide of any of paragraphs 27-31 further comprising atleast 95% identity or similarity to SEQ ID NO: 5.

33. The polynucleotide of any of paragraphs 27-32 further comprising atleast 97% identity or similarity to SEQ ID NO: 5.

34. The polynucleotide of any of paragraphs 27-33 further comprising atleast 99% identity or similarity to SEQ ID NO: 5.

35. A polypeptide comprising an amino acid sequence having at least 95%identity or similarity to SEQ ID NO:6, wherein the polypeptide furthercomprises a tryptophan to a stop mutation at amino acid position 432 ofSEQ ID NO: 6.

36. The polypeptide of paragraph 35 further comprising an amino acidsequence having at least 97% sequence identity or similarity to SEQ IDNO:6.

37. The polypeptide of any of paragraphs 35-36 further comprising anamino acid sequence having at least 99% sequence identity or similarityto SEQ ID NO:6.

38. The polypeptide of any of paragraphs 35-37 comprising an amino acidsequence of SEQ ID NO:6 with a tryptophan to a stop mutation at aminoacid position 432 or a fragment thereof having starch branching enzymeactivity.

39. The polypeptide of any of paragraphs 35-38 comprising an amino acidsequence of SEQ ID NO:6 with a tryptophan to a stop mutation at aminoacid position 432.

40. A polynucleotide encoding an SBEIIa polypeptide comprising atryptophan to a stop mutation at an amino acid corresponding to aminoacid position 446 of SEQ ID NO: 4.

41. The polynucleotide of paragraph 40, wherein the SBEIIa polypeptidefurther comprises an amino acid sequence having at least 95% identity orsimilarity to SEQ ID NO: 4.

42. The polynucleotide of any of paragraphs 40-41, wherein the SBEIIapolypeptide further comprises an amino acid sequence having at least 97%identity or similarity to SEQ ID NO: 4.

43. The polynucleotide of any of paragraphs 40-42, wherein the SBEIIapolypeptide further comprises an amino acid sequence having at least 99%identity or similarity to SEQ ID NO: 4.

44. The polynucleotide of any of paragraphs 40-43 comprising a guanineto adenine mutation at a nucleotide position corresponding to nucleotideposition 5069 of SEQ ID NO: 3.

45. The polynucleotide of any of paragraphs 40-44 further comprising atleast 95% identity or similarity to SEQ ID NO: 3.

46. The polynucleotide of any of paragraphs 40-45 further comprising atleast 97% identity or similarity to SEQ ID NO: 3.

47. The polynucleotide of any of paragraphs 40-46 further comprising atleast 99% identity or similarity to SEQ ID NO: 3.

48. A polypeptide comprising an amino acid sequence having at least 95%identity or similarity to SEQ ID NO:4, wherein the polypeptide furthercomprises a tryptophan to a stop mutation at amino acid position 446 ofSEQ ID NO: 4.

49. The polypeptide of paragraph 48 further comprising an amino acidsequence having at least 97% sequence identity or similarity to SEQ IDNO:4.

50. The polypeptide of paragraphs 48-49 further comprising an amino acidsequence having at least 99% sequence identity or similarity to SEQ IDNO:4.

51. The polypeptide of any of paragraphs 48-50 comprising an amino acidsequence of SEQ ID NO:4 with a tryptophan to a stop mutation at aminoacid position 446 or a fragment thereof having starch branching enzymeactivity.

52. The polypeptide of any of paragraphs 48-51 comprising an amino acidsequence of SEQ ID NO:4 with a tryptophan to a stop mutation at aminoacid position 446.

53. An SBEIIa polynucleotide comprising a guanine to adenine mutation ata nucleotide position corresponding to nucleotide position 5073 of SEQID NO: 3.

54. The polynucleotide of paragraph 53 further comprising at least 95%identity or similarity to SEQ ID NO: 3.

55. The polynucleotide of any of paragraph 53-54 further comprising atleast 97% identity or similarity to SEQ ID NO: 3.

56. The polynucleotide of any of paragraphs 53-55 further comprising atleast 99% identity or similarity to SEQ ID NO: 3.

57. A polynucleotide encoding an SBEIIa polypeptide comprising a glycineto a glutamate mutation at an amino acid corresponding to amino acidposition 467 of SEQ ID NO: 4.

58. The polynucleotide of paragraph 57, wherein the SBEIIa polypeptidefurther comprises an amino acid sequence having at least 95% identity orsimilarity to SEQ ID NO: 4.

59. The polynucleotide of any of paragraphs 57-58, wherein the SBEIIapolypeptide further comprises an amino acid sequence having at least 97%identity or similarity to SEQ ID NO: 4.

60. The polynucleotide of any of paragraphs 57-59, wherein the SBEIIapolypeptide further comprises an amino acid sequence having at least 99%identity or similarity to SEQ ID NO: 4.

61. The polynucleotide of any of paragraphs 57-60 comprising a guanineto adenine mutation at a nucleotide position corresponding to nucleotideposition 5219 of SEQ ID NO: 3.

62. The polynucleotide of any of paragraphs 57-61 further comprising atleast 95% identity or similarity to SEQ ID NO: 3.

63. The polynucleotide of any of paragraphs 57-62 further comprising atleast 97% identity or similarity to SEQ ID NO: 3.

64. The polynucleotide of any of paragraphs 57-63 further comprising atleast 99% identity or similarity to SEQ ID NO: 3.

65. A polypeptide comprising an amino acid sequence having at least 95%identity or similarity to SEQ ID NO:4, wherein the polypeptide furthercomprises a glycine to a glutamate mutation at amino acid position 467of SEQ ID NO: 4.

66. The polypeptide of paragraph 65 further comprising an amino acidsequence having at least 97% sequence identity or similarity to SEQ IDNO:4.

67. The polypeptide of any of paragraphs 65-66 further comprising anamino acid sequence having at least 99% sequence identity or similarityto SEQ ID NO:4.

68. The polypeptide of any of paragraphs 65-67 comprising an amino acidsequence of SEQ ID NO:4 with a glycine to a glutamate mutation at aminoacid position 467 or a fragment thereof having starch branching enzymeactivity.

69. The polypeptide of any of paragraphs 65-68 comprising an amino acidsequence of SEQ ID NO:4 with a glycine to a glutamate mutation at aminoacid position 467.

70. A polynucleotide encoding an SBEIIa polypeptide comprising atryptophan to a stop mutation at an amino acid corresponding to aminoacid position 442 of SEQ ID NO: 6.

71. The polynucleotide of paragraph 70, wherein the SBEIIa polypeptidefurther comprises an amino acid sequence having at least 95% identity orsimilarity to SEQ ID NO: 6.

72. The polynucleotide of any of paragraphs 70-71, wherein the SBEIIapolypeptide further comprises an amino acid sequence having at least 97%identity or similarity to SEQ ID NO: 6.

73. The polynucleotide of any of paragraphs 70-72, wherein the SBEIIapolypeptide further comprises an amino acid sequence having at least 99%identity or similarity to SEQ ID NO: 6.

74. The polynucleotide of any of paragraphs 70-73 comprising a guanineto adenine mutation at a nucleotide position corresponding to nucleotideposition 6335 of SEQ ID NO: 5.

75. The polynucleotide of any of paragraphs 70-74 further comprising atleast 95% identity or similarity to SEQ ID NO: 5.

76. The polynucleotide of any of paragraphs 70-75 further comprising atleast 97% identity or similarity to SEQ ID NO: 5.

77. The polynucleotide of any of paragraphs 70-76 further comprising atleast 99% identity or similarity to SEQ ID NO: 5.

78. A polypeptide comprising an amino acid sequence having at least 95%identity or similarity to SEQ ID NO:6, wherein the polypeptide furthercomprises a tryptophan to a stop mutation at amino acid position 442 ofSEQ ID NO: 6.

79. The polypeptide of paragraph 78 further comprising an amino acidsequence having at least 97% sequence identity or similarity to SEQ IDNO:6.

80. The polypeptide of any of paragraphs 78-79 further comprising anamino acid sequence having at least 99% sequence identity or similarityto SEQ ID NO:6.

81. The polypeptide of any of paragraphs 78-80 further comprising anamino acid sequence of SEQ ID NO:6 with a tryptophan to a stop mutationat amino acid position 442 or a fragment thereof having starch branchingenzyme activity.

82. The polypeptide of any of paragraphs 78-81 comprising an amino acidsequence of SEQ ID NO:6 with a tryptophan to a stop mutation at aminoacid position 442.

83. A polynucleotide encoding an SBEIIb polypeptide comprising atryptophan to a stop mutation at an amino acid corresponding to aminoacid position 285 of SEQ ID NO: 8.

84. The polynucleotide of paragraph 83, wherein the SBEIIb polypeptidefurther comprises an amino acid sequence having at least 95% identity orsimilarity to SEQ ID NO: 8.

85. The polynucleotide of any of paragraphs 83-84, wherein the SBEIIbpolypeptide further comprises an amino acid sequence having at least 97%identity or similarity to SEQ ID NO: 8.

86. The polynucleotide of any of paragraphs 83-85, wherein the SBEIIbpolypeptide further comprises an amino acid sequence having at least 99%identity or similarity to SEQ ID NO: 8.

87. The polynucleotide of any of paragraphs 83-86 comprising a guanineto adenine mutation at a nucleotide position corresponding to nucleotideposition 2282 of SEQ ID NO: 7.

88. The polynucleotide of any of paragraphs 83-87 further comprising atleast 95% identity or similarity to SEQ ID NO: 7.

89. The polynucleotide of any of paragraphs 83-88 further comprising atleast 97% identity or similarity to SEQ ID NO: 7.

90. The polynucleotide of any of paragraphs 83-89 further comprising atleast 99% identity or similarity to SEQ ID NO: 7.

91. A polypeptide comprising an amino acid sequence having at least 95%identity or similarity to SEQ ID NO:8, wherein the polypeptide furthercomprises a tryptophan to a stop mutation at amino acid position 285 ofSEQ ID NO: 8.

92. The polypeptide of paragraph 91 further comprising an amino acidsequence having at least 97% sequence identity or similarity to SEQ IDNO:8.

93. The polypeptide of any of paragraphs 91-92 further comprising anamino acid sequence having at least 99% sequence identity or similarityto SEQ ID NO:8.

94. The polypeptide of any of paragraphs 91-93 further comprising anamino acid sequence of SEQ ID NO:8 with a tryptophan to a stop mutationat amino acid position 285 or a fragment thereof having starch branchingenzyme activity.

95. The polypeptide of any of paragraphs 91-94 comprising an amino acidsequence of SEQ ID NO:8 with a tryptophan to a stop mutation at aminoacid position 285.

96. A wheat plant comprising a polynucleotide of any of paragraphs 1-8,14-21, 27-34, 40-47, 53-56, 57-64, 70-77, and 83-90.

97. A wheat plant comprising at least two non-transgenic mutations in anSBEII gene, wherein at least one mutation is in the SBEIIa gene asrecited in any of paragraphs 1-8, 14-21, 27-34, 40-47, 53-56, 57-64, and70-77.

98. The wheat plant of any of paragraphs 96-97, wherein a secondnon-transgenic mutation is in the SBEIIb gene. The SBEIIb mutations maybe as recited in paragraphs 83-90.

99. The wheat plant of any of paragraphs 96-98, wherein the first andsecond mutations are in the SBEIIa gene.

100. The wheat plant of any of paragraphs 96-99, wherein the first andsecond mutations are in the same genome.

101. The wheat plant of any of paragraphs 96-100, wherein the first andsecond mutations are in different genomes.

102. The wheat plant of any of paragraphs 96-101, further comprising atleast three non-transgenic mutations in the SBEII gene.

103. The wheat plant of any of paragraphs 96-102, wherein two mutationsare in the same genome.

104. The wheat plant of any of paragraphs 96-103, wherein threemutations are in different genomes.

105. The wheat plant of any of paragraphs 96-104, wherein the threemutations are in each of the A genome, B genome and D genome. Any numberof mutations are possible including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, andgreater than 10 mutations in the SBEIIa gene and including 1, 2, 3, 4,5, 6, 7, 8, 9, 10, and greater than 10 mutations in the SBEIIb gene.

106. A wheat plant comprising at least two polynucleotides as recited inany of paragraphs 1-8, 14-21, 27-34, 40-47, 53-56, 57-64, 70-77, and83-90

107. A wheat plant comprising a polypeptide of any of paragraphs 9-13,22-26, 35-39, 48-52, 65-69, 78-82, and 91-95.

108. The wheat plant of any of paragraphs 96-107, wherein the wheat isdiploid, tetraploid or hexaploid.

109. A hexaploid wheat plant comprising at least one mutation in eachSBEIIa gene, wherein the mutation in the SBEIIa gene of the A genomecorresponds to a guanine to adenine mutation at nucleotide position 5267of SEQ ID NO: 1, wherein the mutation in the SBEIIa gene of the B genomecorresponds to a guanine to adenine mutation at nucleotide position 5308of SEQ ID NO: 3; and wherein the mutation in the SBEIIa gene of the Dgenome corresponds to a guanine to adenine mutation at nucleotideposition 6305 of SEQ ID NO: 5.

110. A hexaploid wheat plant comprising at least one mutation in eachSBEIIa gene, wherein the mutation in the SBEIIa gene of the A genomecorresponds to a guanine to adenine mutation at nucleotide position 5267of SEQ ID NO: 1, wherein the mutation in the SBEIIa gene of the B genomecorresponds to a guanine to adenine mutation at nucleotide position 5069of SEQ ID NO: 3; and wherein the mutation in the SBEIIa gene of the Dgenome corresponds to a guanine to adenine mutation at nucleotideposition 6335 of SEQ ID NO: 5.

111. A hexaploid wheat plant comprising at least one mutation in eachSBEIIa gene, wherein the mutation in the SBEIIa gene of the A genomecorresponds to a guanine to adenine mutation at nucleotide position 5267of SEQ ID NO: 1, wherein the mutation in the SBEIIa gene of the B genomecorresponds to a guanine to adenine mutation at nucleotide position 5193of SEQ ID NO: 3; and wherein the mutation in the SBEIIa gene of the Dgenome corresponds to a guanine to adenine mutation at nucleotideposition 6305 of SEQ ID NO: 5.

112. A wheat plant comprising at least one mutation in each SBEIIa gene,wherein the mutation in the SBEIIa gene of the A genome corresponds to aguanine to adenine mutation at nucleotide position 5267 of SEQ ID NO: 1,wherein the mutation in the SBEIIa gene of the B genome corresponds to aguanine to adenine mutation at nucleotide position 5073 of SEQ ID NO: 3.

113. A wheat plant comprising at least one mutation in each SBEIIa gene,wherein the mutation in the SBEIIa gene of the A genome corresponds to aguanine to adenine mutation at nucleotide position 5267 of SEQ ID NO: 1,wherein the mutation in the SBEIIa gene of the B genome corresponds to aguanine to adenine mutation at nucleotide position 5219 of SEQ ID NO: 3.

114. A wheat plant comprising at least one mutation in each SBEIIa gene,wherein the mutation in the SBEIIa gene of the A genome corresponds to aguanine to adenine mutation at nucleotide position 5267 of SEQ ID NO: 1,wherein the mutation in the SBEIIa gene of the B genome corresponds to aguanine to adenine mutation at nucleotide position 5033 of SEQ ID NO: 3.

115. A wheat seed comprising a polynucleotide of any of paragraphs 1-8,14-21, 27-34, 40-47, 53-56, 57-64, 70-77, and 83-90.

116. A wheat seed comprising at least two non-transgenic mutations in anSBEII gene, wherein at least one mutation is in the SBEIIa gene asrecited in any of paragraphs 1-8, 14-21, 27-34, 40-47, 53-56, 57-64,70-77, and 83-90.

117. The wheat seed of any of paragraphs 115-115, wherein a secondnon-transgenic mutation is in the SBEIIb gene.

118. The wheat seed of any of paragraphs 115-117, wherein the first andsecond mutations are in the SBEIIa gene.

119. The wheat seed of any of paragraphs 115-118, wherein the first andsecond mutations are in the same genome.

120. The wheat seed of any of paragraphs 115-119, wherein the first andsecond mutations are in different genomes.

121. The wheat seed of any of paragraphs 115-120 further comprising atleast three non-transgenic mutations in the SBEII gene.

122. The wheat seed of any of paragraphs 115-121, wherein threemutations are in the same genome.

123. The wheat seed of any of paragraphs 115-122, wherein threemutations are in different genomes.

124. The wheat seed of any of paragraphs 115-123, wherein the threemutations are in each of the A genome, B genome and D genome.

125. A wheat seed comprising at least two polynucleotides as recited inany of paragraphs 1-8, 14-21, 27-34, 40-47, 53-56, 57-64, 70-77, and83-90.

126. A wheat seed comprising a polypeptide of any of paragraphs 9-13,22-26, 35-39, 48-52, 65-69, 78-82, and 91-95.

127. The wheat seed of any of paragraphs 115-126, wherein the wheat isdiploid, tetraploid or hexaploid.

128. A hexaploid wheat seed comprising at least one mutation in eachSBEIIa gene, wherein the mutation in the SBEIIa gene of the A genomecorresponds to a guanine to adenine mutation at nucleotide position 5267of SEQ ID NO: 1, wherein the mutation in the SBEIIa gene of the B genomecorresponds to a guanine to adenine mutation at nucleotide position 5308of SEQ ID NO: 3; and wherein the mutation in the SBEIIa gene of the Dgenome corresponds to a guanine to adenine mutation at nucleotideposition 6305 of SEQ ID NO: 5.

129. A hexaploid wheat seed comprising at least one mutation in eachSBEIIa gene, wherein the mutation in the SBEIIa gene of the A genomecorresponds to a guanine to adenine mutation at nucleotide position 5267of SEQ ID NO: 1, wherein the mutation in the SBEIIa gene of the B genomecorresponds to a guanine to adenine mutation at nucleotide position 5069of SEQ ID NO: 3; and wherein the mutation in the SBEIIa gene of the Dgenome corresponds to a guanine to adenine mutation at nucleotideposition 6335 of SEQ ID NO: 5.

130. A hexaploid wheat seed comprising at least one mutation in eachSBEIIa gene, wherein the mutation in the SBEIIa gene of the A genomecorresponds to a guanine to adenine mutation at nucleotide position 5267of SEQ ID NO: 1, wherein the mutation in the SBEIIa gene of the B genomecorresponds to a guanine to adenine mutation at nucleotide position 5193of SEQ ID NO: 3; and wherein the mutation in the SBEIIa gene of the Dgenome corresponds to a guanine to adenine mutation at nucleotideposition 6305 of SEQ ID NO: 5.

131. A wheat seed comprising at least one mutation in each SBEIIa gene,wherein the mutation in the SBEIIa gene of the A genome corresponds to aguanine to adenine mutation at nucleotide position 5267 of SEQ ID NO: 1,wherein the mutation in the SBEIIa gene of the B genome corresponds to aguanine to adenine mutation at nucleotide position 5073 of SEQ ID NO: 3.

132. A wheat seed comprising at least one mutation in each SBEIIa gene,wherein the mutation in the SBEIIa gene of the A genome corresponds to aguanine to adenine mutation at nucleotide position 5267 of SEQ ID NO: 1,wherein the mutation in the SBEIIa gene of the B genome corresponds to aguanine to adenine mutation at nucleotide position 5219 of SEQ ID NO: 3.

133. A wheat seed comprising at least one mutation in each SBEIIa gene,wherein the mutation in the SBEIIa gene of the A genome corresponds to aguanine to adenine mutation at nucleotide position 5267 of SEQ ID NO: 1,wherein the mutation in the SBEIIa gene of the B genome corresponds to aguanine to adenine mutation at nucleotide position 5033 of SEQ ID NO: 3.

134. Wheat grain comprising a polynucleotide of any of paragraphs 1-8,14-21, 27-34, 40-47, 53-56, 57-64, 70-77, and 83-90.

135. Wheat grain comprising at least two non-transgenic mutations in anSBEII gene, wherein one mutation is in the SBEIIa gene as recited in anyof paragraphs 1-8, 14-21, 27-34, 40-47, 53-56, 57-64, 70-77, and 83-90.

136. The wheat grain of any of paragraphs 134-135, wherein a secondnon-transgenic mutation is in the SBEIIb gene.

137. The wheat grain of any of paragraphs 134-136, wherein the first andsecond mutations are in the SBEIIa gene.

138. The wheat grain of any of paragraphs 134-137, wherein the first andsecond mutations are in the same genome.

139. The wheat grain of any of paragraphs 134-138, wherein the first andsecond mutations are in different genomes.

140. The wheat grain of any of paragraphs 134-139, further comprising atleast three non-transgenic mutations in the SBEII gene.

141 The wheat grain of any of paragraphs 134-140, wherein the threemutations are in the same genome.

142. The wheat grain of any of paragraphs 134-141, wherein the threemutations are in different genomes.

143. The wheat grain of any of paragraphs 134-142, wherein the threemutations are in each of the A genome, B genome and D genome.

144. Wheat grain comprising at least two polynucleotides as recited inany of paragraphs 1-8, 14-21, 27-34, 40-47, 53-56, 57-64, 70-77, and83-90.

145. Wheat grain comprising a polypeptide of any of paragraphs 9-13,22-26, 35-39, 48-52, 65-69, 78-82, and 91-95.

146. Wheat grain of any of paragraphs 134-145, wherein the wheat isdiploid, tetraploid or hexaploid.

147. A hexaploid wheat grain comprising at least one mutation in eachSBEIIa gene, wherein the mutation in the SBEIIa gene of the A genomecorresponds to a guanine to adenine mutation at nucleotide position 5267of SEQ ID NO: 1, wherein the mutation in the SBEIIa gene of the B genomecorresponds to a guanine to adenine mutation at nucleotide position 5308of SEQ ID NO: 3; and wherein the mutation in the SBEIIa gene of the Dgenome corresponds to a guanine to adenine mutation at nucleotideposition 6305 of SEQ ID NO: 5.

148. A hexaploid wheat grain comprising at least one mutation in eachSBEIIa gene, wherein the mutation in the SBEIIa gene of the A genomecorresponds to a guanine to adenine mutation at nucleotide position 5267of SEQ ID NO: 1, wherein the mutation in the SBEIIa gene of the B genomecorresponds to a guanine to adenine mutation at nucleotide position 5069of SEQ ID NO: 3; and wherein the mutation in the SBEIIa gene of the Dgenome corresponds to a guanine to adenine mutation at nucleotideposition 6335 of SEQ ID NO: 5.

149. A hexaploid wheat grain comprising at least one mutation in eachSBEIIa gene, wherein the mutation in the SBEIIa gene of the A genomecorresponds to a guanine to adenine mutation at nucleotide position 5267of SEQ ID NO: 1, wherein the mutation in the SBEIIa gene of the B genomecorresponds to a guanine to adenine mutation at nucleotide position 5193of SEQ ID NO: 3; and wherein the mutation in the SBEIIa gene of the Dgenome corresponds to a guanine to adenine mutation at nucleotideposition 6305 of SEQ ID NO: 5.

150. A wheat grain comprising at least one mutation in each SBEIIa gene,wherein the mutation in the SBEIIa gene of the A genome corresponds to aguanine to adenine mutation at nucleotide position 5267 of SEQ ID NO: 1,wherein the mutation in the SBEIIa gene of the B genome corresponds to aguanine to adenine mutation at nucleotide position 5073 of SEQ ID NO: 3.

151. A wheat grain comprising at least one mutation in each SBEIIa gene,wherein the mutation in the SBEIIa gene of the A genome corresponds to aguanine to adenine mutation at nucleotide position 5267 of SEQ ID NO: 1,wherein the mutation in the SBEIIa gene of the B genome corresponds to aguanine to adenine mutation at nucleotide position 5219 of SEQ ID NO: 3.

152. A wheat grain comprising at least one mutation in each SBEIIa gene,wherein the mutation in the SBEIIa gene of the A genome corresponds to aguanine to adenine mutation at nucleotide position 5267 of SEQ ID NO: 1,wherein the mutation in the SBEIIa gene of the B genome corresponds to aguanine to adenine mutation at nucleotide position 5033 of SEQ ID NO: 3.

153. Wheat flour comprising a polynucleotide of any of paragraphs 1-8,14-21, 27-34, 40-47, 53-56, 57-64, 70-77, and 83-90.

154. Wheat flour comprising at least two non-transgenic mutations in anSBEII gene, wherein one mutation is in the SBEIIa gene as recited in anyof paragraphs 1-8, 14-21, 27-34, 40-47, 53-56, 57-64, 70-77, and 83-90.

155. The wheat flour of any of paragraphs 153-154, wherein a secondnon-transgenic mutation is in the SBEIIb gene.

156. The wheat flour of any of paragraphs 153-155, wherein the first andsecond mutations are in the SBEIIa gene.

157. The wheat flour of any of paragraphs 153-156, wherein the first andsecond mutations are in the same genome.

158. The wheat flour of any of paragraphs 153-157, wherein the first andsecond mutations are in different genomes.

159. The wheat flour of any of paragraphs 153-158, further comprising atleast three non-transgenic mutations in the SBEII gene.

160. The wheat flour of any of paragraphs 153-159, wherein the threemutations are in the same genome.

161. The wheat flour of any of paragraphs 153-160, wherein the threemutations are in different genomes.

162. The wheat flour of any of paragraphs 153-161, wherein the threemutations are in each of the A genome, B genome and D genome.

163. Wheat flour comprising at least two polynucleotides as recited inany of paragraphs 1-8, 14-21, 27-34, 40-47, 53-56, 57-64, 70-77, and83-90.

164. Wheat flour comprising a polypeptide of any of paragraphs 9-13,22-26, 35-39, 48-52, 65-69, 78-82, and 91-95.

165. Wheat flour of any of paragraphs 153-164, wherein the wheat isdiploid, tetraploid or hexaploid.

166. A hexaploid wheat flour comprising at least one mutation in eachSBEIIa gene, wherein the mutation in the SBEIIa gene of the A genomecorresponds to a guanine to adenine mutation at nucleotide position 5267of SEQ ID NO: 1, wherein the mutation in the SBEIIa gene of the B genomecorresponds to a guanine to adenine mutation at nucleotide position 5308of SEQ ID NO: 3; and wherein the mutation in the SBEIIa gene of the Dgenome corresponds to a guanine to adenine mutation at nucleotideposition 6305 of SEQ ID NO: 5.

167. A hexaploid wheat flour comprising at least one mutation in eachSBEIIa gene, wherein the mutation in the SBEIIa gene of the A genomecorresponds to a guanine to adenine mutation at nucleotide position 5267of SEQ ID NO: 1, wherein the mutation in the SBEIIa gene of the B genomecorresponds to a guanine to adenine mutation at nucleotide position 5069of SEQ ID NO: 3; and wherein the mutation in the SBEIIa gene of the Dgenome corresponds to a guanine to adenine mutation at nucleotideposition 6335 of SEQ ID NO: 5.

168. A hexaploid wheat flour comprising at least one mutation in eachSBEIIa gene, wherein the mutation in the SBEIIa gene of the A genomecorresponds to a guanine to adenine mutation at nucleotide position 5267of SEQ ID NO: 1, wherein the mutation in the SBEIIa gene of the B genomecorresponds to a guanine to adenine mutation at nucleotide position 5193of SEQ ID NO: 3; and wherein the mutation in the SBEIIa gene of the Dgenome corresponds to a guanine to adenine mutation at nucleotideposition 6305 of SEQ ID NO: 5.

169. A wheat flour comprising at least one mutation in each SBEIIa gene,wherein the mutation in the SBEIIa gene of the A genome corresponds to aguanine to adenine mutation at nucleotide position 5267 of SEQ ID NO: 1,wherein the mutation in the SBEIIa gene of the B genome corresponds to aguanine to adenine mutation at nucleotide position 5073 of SEQ ID NO: 3.

170. A wheat flour comprising at least one mutation in each SBEIIa gene,wherein the mutation in the SBEIIa gene of the A genome corresponds to aguanine to adenine mutation at nucleotide position 5267 of SEQ ID NO: 1,wherein the mutation in the SBEIIa gene of the B genome corresponds to aguanine to adenine mutation at nucleotide position 5219 of SEQ ID NO: 3.

171. A wheat flour comprising at least one mutation in each SBEIIa gene,wherein the mutation in the SBEIIa gene of the A genome corresponds to aguanine to adenine mutation at nucleotide position 5267 of SEQ ID NO: 1,wherein the mutation in the SBEIIa gene of the B genome corresponds to aguanine to adenine mutation at nucleotide position 5033 of SEQ ID NO: 3.

172. A food product comprising the wheat grain of any of paragraphs134-152.

173. A food product comprising the wheat flour of any of paragraphs153-171.

174. Use of a polynucleotide according to any of paragraphs 1-8, 14-21,27-34, 40-47, 53-56, 57-64, 70-77, and 83-90 for the production of wheathaving increased amylose levels compared to wild type wheat, whereinsaid polynucleotide contributes to the increased amylose levels.

175. Use of a polynucleotide according to any of paragraphs 1-8, 14-21,27-34, 40-47, 53-56, 57-64, 70-77, and 83-90 for the selection of wheathaving increased amylose levels compared to wild type wheat, whereingenomic DNA is isolated from the wheat and a segment of said SBEII geneis amplified and the presence of said gene is detected.

176. Use of a polypeptide according to any of paragraphs 9-13, 22-26,35-39, 48-52, 65-69, 78-82, and 91-95 for the production of wheat havingincreased amylose levels compared to wild type wheat, wherein saidpolynucleotide contributes to the increased amylose levels.

177. Use of a polypeptide according to any of paragraphs 9-13, 22-26,35-39, 48-52, 65-69, 78-82, and 91-95 for the selection of wheat havingincreased amylose levels compared to wild type wheat, wherein genomicDNA is isolated from the wheat and a segment of said SBEII gene isamplified and the presence of said gene is detected.

Example 1

Mutagenesis

In accordance with one exemplary embodiment of the present invention,wheat seeds of the hexaploid cultivar (Triticum aestivum) Express and ofthe tetraploid cultivar (Triticum turgidum, Durum) Kronos were vacuuminfiltrated in H₂O (approximately 1,000 seeds/100 ml H₂O forapproximately 4 minutes). The seeds were then placed on a shaker (45rpm) in a fume hood at room temperature. The mutagen ethylmethanesulfonate (EMS) was added to the imbibing seeds to finalconcentrations ranging from about 0.75% to about 1.2% (v/v). Followingan 18-hour incubation period, the EMS solution was replaced 4 times withfresh H₂O. The seeds were then rinsed under running water for about 4-8hours. Finally, the mutagenized seeds were planted (96/tray) in pottingsoil and allowed to germinate indoors. Plants that were four to sixweeks old were transferred to the field to grow to fully mature M1plants. The mature M1 plants were allowed to self-pollinate and thenseeds from the M1 plant were collected and planted to produce M2 plants.

DNA Preparation

DNA from the M2 plants produced in accordance with the above descriptionwas extracted and prepared in order to identify which M2 plants carrieda mutation at one or more of their SBEII loci. The M2 plant DNA wasprepared using the methods and reagents contained in the Qiagen®(Valencia, Calif.) DNeasy® 96 Plant Kit. Approximately 50 mg of frozenplant sample was placed in a sample tube with a tungsten bead, frozen inliquid nitrogen and ground 2 times for 1 minute each at 20 Hz using theRetsch® Mixer Mill MM 300. Next, 400 μl of solution AP1 [Buffer AP1,solution DX and RNAse (100 mg/ml)] at 80° C. was added to the sample.The tube was sealed and shaken for 15 seconds. Following the addition of130 μl Buffer AP2, the tube was shaken for 15 seconds. The samples wereplaced in a freezer at minus 20° C. for at least 1 hour. The sampleswere then centrifuged for 20 minutes at 5,600×g. A 400 μl aliquot ofsupernatant was transferred to another sample tube. Following theaddition of 600 μl of Buffer AP3/E, this sample tube was capped andshaken for 15 seconds. A filter plate was placed on a square well blockand 1 ml of the sample solution was applied to each well and the platewas sealed. The plate and block were centrifuged for 4 minutes at5,600×g. Next, 800 μl of Buffer AW was added to each well of the filterplate, sealed and spun for 15 minutes at 5,600×g in the square wellblock. The filter plate was then placed on a new set of sample tubes and80 μl of Buffer AE was applied to the filter. It was capped andincubated at room temperature for 1 minute and then spun for 2 minutesat 5600×g. This step was repeated with an additional 80 μl Buffer AE.The filter plate was removed and the tubes containing the pooledfiltrates were capped. The individual samples were then normalized to aDNA concentration of 5 to 10 ng/μl.

Tilling

The M2 DNA was pooled into groups of two individual plants. The DNAconcentration for each individual within the pool was approximately 0.8ng/μl with a final concentration of 1.6 ng/μl for the entire pool. Then,5 μl of the pooled DNA samples (or 8 ng wheat DNA) was arrayed onmicrotiter plates and subjected to gene-specific PCR.

PCR amplification was performed in 15 μl volumes containing 2.5 ngpooled DNA, 0.75X ExTaq buffer (Panvera®, Madison, Wis.), 2.6 mM MgCl₂,0.3 mM dNTPs, 0.3 μM primers, and 0.05U Ex-Taq (Panvera®) DNApolymerase. PCR amplification was performed using an MJ Research®thermal cycler as follows: 95° C. for 2 minutes; 8 cycles of “touchdownPCR” (94° C. for 20 second, followed by annealing step starting at70-68° C. for 30 seconds and decreasing 1° C. per cycle, then atemperature ramp of 0.5° C. per second to 72° C. followed by 72° C. for1 minute); 25-45 cycles of 94° C. for 20 seconds, 63-61° C. for 30seconds, ramp 0.5° C./sec to 72° C., 72° C. for 1 minute; 72° C. for 8minutes; 98° C. for 8 minutes; 80° C. for 20 seconds; 60 cycles of 80°C. for 7 seconds—0.3 degrees/cycle.

The PCR primers (MWG Biotech, Inc., High Point, N.C.) were mixed asfollows:

2.5 μl 100 μM IRD-700 labeled left primer

7.5 μl 100 μM left primer

9.0 μl 100 μM IRD-800 labeled right primer

1.0 μl 100 μM right primer

A label can be attached to each primer as described or to only one ofthe primers. Alternatively, Cy5.5 modified primers could be used. Thelabel was coupled to the oligonucleotide using conventionalphosphoramidite chemistry.

PCR products (15 μl) were digested in 96-well plates. Next, 30 μl of asolution containing 10 mM HEPES[4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid] (pH 7.5), 10 mMMgSO₄, 0.002% (w/v) Triton® X-100, 20 ng/ml of bovine serum albumin, andSurveyor® endonuclease (Transgenomic®, Inc.; 1:100,000 dilution) wasadded with mixing on ice, and the plate was incubated at 45° C. for 15minutes. The specific activity of the Surveyor enzyme was 800 units/μl,where a unit was defined by the manufacturer as the amount of enzymerequired to produce 1 ng of acid-soluble material from sheared, heatdenatured calf thymus DNA at pH 8.5 in one minute at 37° C. Reactionswere stopped by addition of 10 μl of a 2.5 M NaCl solution with 0.5mg/ml blue dextran and 75 mM EDTA, followed by the addition of 80 μlisopropanol. The reactions were precipitated at room temperature, spunat 4,000 rpm for 30 minutes in an Eppendorf Centrifuge 5810. Pelletswere resuspended in 8 μl of 33% formamide with 0.017% bromophenol bluedye, heated at 80° C. for 7 minutes and then at 95° C. for 2 minutes.Samples were transferred to a membrane comb using a comb-loading robot(MWG Biotech). The comb was inserted into a slab acrylamide gel (6.5%),electrophoresed for 10 min, and removed. Electrophoresis was continuedfor 4 hours at 1,500-V, 40-W, and 40-mA limits at 50° C.

During electrophoresis, the gel was imaged using a LI-COR® (Lincoln,Nebr.) scanner which was set at a channel capable of detecting the IRDye 700 and 800 labels. The gel image showed sequence-specific patternof background bands common to all 96 lanes. Rare events, such asmutations, create new bands that stand out above the background pattern.Plants with bands indicative of mutations of interest were evaluated byTILLING individual members of a pool mixed with wild type DNA and thensequencing individual PCR products. Plants carrying mutations confirmedby sequencing were grown up as described above (e.g., the M2 plant couldbe backcrossed or outcrossed twice in order to eliminate backgroundmutations and self-pollinated in order to create a plant that washomozygous for the mutation) or crossed to another plant containingSBEII mutations in a different homoeolog.

Plants that were identified with severe mutations in SBEIIa of the A, B,or D genome were crossed with other plants that contained severemutations in SBEIIa in other genomes. Severe mutations included thosemutations that were predicted to have a deleterious effect on proteinfunction by their SIFT and PSSM, as well as those mutations thatresulted in the introduction of a stop codon (truncation mutation) or amutation at a splice junction. Table 8 shows examples of crosses thatwere made.

With regard to Tables 8-12, nucleic acid designations of the mutationsin SBEIIa of the A genome correspond to the position in the referencesequence SEQ ID NO: 1. Amino acid designations of the SBEIIa polypeptideof the A genome correspond to the amino acid position of referencesequence SEQ ID NO: 2. Nucleic acid designations of the mutations inSBEIIa of the B genome correspond to the position in the referencesequence SEQ ID NO: 3. Amino acid designations of the SBEIIa polypeptideof the B genome correspond to the amino acid position of referencesequence SEQ ID NO: 4. Nucleic acid designations of the mutations inSBEIIa of the D genome correspond to the position in the referencesequence SEQ ID NO: 5. Amino acid designations of the SBEIIa polypeptideof the A genome correspond to the amino acid position of referencesequence SEQ ID NO: 6. Nucleic acid designations of the mutations inSBEIIb of the A genome correspond to the position in the referencesequence SEQ ID NO: 7. Amino acid designations of the SBEIIb polypeptideof the A genome correspond to the amino acid position of referencesequence SEQ ID NO: 8. Nucleic acid designations of the mutations inSBEIIb of the B genome correspond to the position in the referencesequence SEQ ID NO: 9. Amino acid designations of the SBEIIb polypeptideof the B genome correspond to the amino acid position of referencesequence SEQ ID NO: 10. Nucleic acid designations of the mutations inSBEIIb of the D genome correspond to the position in the referencesequence SEQ ID NO: 11. Amino acid designations of the SBEIIbpolypeptide of the A genome correspond to the amino acid position ofreference sequence SEQ ID NO: 12.

TABLE 8 Examples of wheat plants identified which had a mutation inSBEIIa that was predicted to be severe and the crosses that were made toplants with severe SBEIIa mutations in a different genome. NucleotideCross Variety Gene Mutation A.A. Mutation 1 Express SBEIIaA G5267A W436*Express SBEIIaB G5038A W436* Express SBEIIaD G6305A W432* 2 ExpressSBEIIaA G5267A W436* Express SBEIIaB G5069A W446* Express SBEIIaD G6335AW442* 3 Express SBEIIaA G5267A W436* Express SBEIIaB G5193A W458*Express SBEIIaD G6305A W432* 4 Kronos SBEIIaA G5267A W436* KronosSBEIIaB G5073A Splice Junction 5 Kronos SBEIIaA G5267A W436* KronosSBEIIaB G5219A G467E 6 Kronos SBEIIaA G5267A W436* Kronos SBEIIaB G5033AW434*

Additionally, Express wheat plants identified as containing mutations inSBEIIa were rescreened for mutations in SBEIIb of the same genome usinghomoeologue specific primers. Plants with mutations in both SBEIIa andSBEIIb of each genome were sequenced and the plants containing severemutations in both linked genes of the same genome were grown up andself-pollinated to obtain homozygous lines and confirm linkage of themutations in SBEIIa and SBEIIb. Plants with mutations in both SBEIIa andSBEIIb in the same genome were crossed to plants with linked SBEIImutations in other genomes to obtain wheat lines with linked mutationsin all three genomes.

TABLE 9: Examples of twelve Express wheat plants identified which hadsevere mutations in both SBEIIa and SBEIIb of the same genome (i.e.,linked mutations) are shown in Table 9. The SBEIIa and SBEIIb genes arelocated close together on the chromosome and mutation segregationstudies showed that these mutations were linked and were not inheritedindependently. It would be obvious to one skilled in the art that analternative approach to identify linked mutations in both genes would beto first identify plants with mutations in their SBEIIb genomes and thenrescreen these individuals for mutations in their SBEIIa genomes. Itwould also be obvious to one skilled in the art that an alternativeapproach to obtain linked mutations in both genes would be to identifyplants in which recombination has occurred between mutations in SBEIIaand SBEIIb.

TABLE 9 Wheat plants with mutations in both SBEIIa and SBEIIb of thesame genome A.A. A.A. Nucleotide Mu- Nucleotide Mu- Plant Gene Mutationtation Gene Mutation tation 1 SBEIIaA C5804T P519S SBEIIbA C2617T P336L2 SBEIIaA G5463A G472E SBEIIbA G2282A W285* 3 SBEIIaA G5463A G472ESBEIIbA G2282A W285* 4 SBEIIaA G5463A G472E SBEIIbA G2282A W285* 5SBEIIaA G2605A G264D SBEIIbA G1356A E216K 6 SBEIIaA C5757T A503V SBEIIbAG278A W59* 7 SBEIIaD G6306A D433N SBEIIbD C4573T R325W 8 SBEIIaD G5156AG374E SBEIIbD C4246T P275L 9 SBEIIaD G5156A G374E SBEIIbD C4246T P275L10 SBEIIaD C3743T S266F SBEIIbD G4290A V290M 11 SBEIIaB G5219A G467ESBEIIbB C3232T R325W 12 SBEIIaB G2386A G233D SBEIIbB C2786T P263LPlants that were homozygous for severe linked mutations (SBEIIa andSBEIIb) in each genome were crossed with plants containing severe linkedmutations in other genomes to create plants that had linked SBEIIa andSBEIIb mutations in all three genomes. Multiple combinations ofmutations within genomes were produced during the crossing.

TABLE 10 Examples of wheat plants identified that had a severe mutationin SBEIIa and SBEIIb of each genome and crosses to achieve plants withmutations in both SBEIIa and SBEIIb of all three genomes. A.A. A.A.Nucleotide Mu- Nucleotide Mu- Cross Gene Mutation tation Gene Mutationtation 1 SBEIIaA G2605A G264D SBEIIbA G1356A E216K SBEIIaB G2386A G233DSBEIIbB C2786T P263L SBEIIaD G6306A D433N SBEIIbD C4573T R325W 2 SBEIIaAG2605A G264D SBEIIbA G1356A E216K SBEIIaB G2386A G233D SBEIIbB C2786TP263L SBEIIaD G5156A G374E SBEIIbD C4246T P275L 3 SBEIIaA G2605A G264DSBEIIbA G1356A E216K SBEIIaB G2386A G233D SBEIIbB C2786T P263L SBEIIaDC3743T S266F SBEIIbD G4290A V290M 4 SBEIIaA C5804T P519S SBEIIbA C2617TP336L SBEIIaB G2386A G233D SBEIIbB C2786T P263L SBEIIaD G6306A D433NSBEIIbD C4573T R325W 5 SBEIIaA C5804T P519S SBEIIbA C2617T P336L SBEIIaBG2386A G233D SBEIIbB C2786T P263L SBEIIaD G5156A G374E SBEIIbD C4246TP275L 6 SBEIIaA C5804T P519S SBEIIbA C2617T P336L SBEIIaB G2386A G233DSBEIIbB C2786T P263L SBEIIaD C3743T S266F SBEIIbD G4290A V290M 7 SBEIIaAG5463A G472E SBEIIbA G2282A W285* SBEIIaB G2386A G233D SBEIIbB C2786TP263L SBEIIaD G6306A D433N SBEIIbD C4573T R325W 8 SBEIIaA G5463A G472ESBEIIbA G2282A W285* SBEIIaB G2386A G233D SBEIIbB C2786T P263L SBEIIaDG5156A G374E SBEIIbD C4246T P275L 9 SBEIIaA G5463A G472E SBEIIbA G2282AW285* SBEIIaB G2386A G233D SBEIIbB C2786T P263L SBEIIaD C3743T S266FSBEIIbD G4290A V290M 10 SBEIIaA C5757T A503V SBEIIbA G278A W59* SBEIIaBG2386A G233D SBEIIbB C2786T P263L SBEIIaD G6306A D433N SBEIIbD C4573TR325W 11 SBEIIaA C5757T A503V SBEIIbA G278A W59* SBEIIaB G2386A G233DSBEIIbB C2786T P263L SBEIIaD G5156A G374E SBEIIbD C4246T P275L 12SBEIIaA C5757T A503V SBEIIbA G278A W59* SBEIIaB G2386A G233D SBEIIbBC2786T P263L SBEIIaD C3743T S266F SBEIIbD G4290A V290M 13 SBEIIaA G2605AG264D SBEIIbA G1356A E216K SBEIIaB G5219A G467E SBEIIbB C3232T R325WSBEIIaD G6306A D433N SBEIIbD C4573T R325W 14 SBEIIaA G2605A G264DSBEIIbA G1356A E216K SBEIIaB G5219A G467E SBEIIbB C3232T R325W SBEIIaDG5156A G374E SBEIIbD C4246T P275L 15 SBEIIaA G2605A G264D SBEIIbA G1356AE216K SBEIIaB G5219A G467E SBEIIbB C3232T R325W SBEIIaD C3743T S266FSBEIIbD G4290A V290M 16 SBEIIaA C5804T P519S SBEIIbA C2617T P336LSBEIIaB G5219A G467E SBEIIbB C3232T R325W SBEIIaD G6306A D433N SBEIIbDC4573T R325W 17 SBEIIaA C5804T P519S SBEIIbA C2617T P336L SBEIIaB G5219AG467E SBEIIbB C3232T R325W SBEIIaD G5156A G374E SBEIIbD C4246T P275L 18SBEIIaA C5804T P519S SBEIIbA C2617T P336L SBEIIaB G5219A G467E SBEIIbBC3232T R325W SBEIIaD C3743T S266F SBEIIbD G4290A V290M 19 SBEIIaA G5463AG472E SBEIIbA G2282A W285* SBEIIaB G5219A G467E SBEIIbB C3232T R325WSBEIIaD G6306A D433N SBEIIbD C4573T R325W 20 SBEIIaA G5463A G472ESBEIIbA G2282A W285* SBEIIaB G5219A G467E SBEIIbB C3232T R325W SBEIIaDG5156A G374E SBEIIbD C4246T P275L 21 SBEIIaA G5463A G472E SBEIIbA G2282AW285* SBEIIaB G5219A G467E SBEIIbB C3232T R325W SBEIIaD C3743T S266FSBEIIbD G4290A V290M 22 SBEIIaA C5757T A503V SBEIIbA G278A W59* SBEIIaBG5219A G467E SBEIIbB C3232T R325W SBEIIaD G6306A D433N SBEIIbD C4573TR325W 23 SBEIIaA C5757T A503V SBEIIbA G278A W59* SBEIIaB G5219A G467ESBEIIbB C3232T R325W SBEIIaD G5156A G374E SBEIIbD C4246T P275L 24SBEIIaA C5757T A503V SBEIIbA G278A W59* SBEIIaB G5219A G467E SBEIIbBC3232T R325W SBEIIaD C3743T S266F SBEIIbD G4290A V290M

TABLE 11 Three examples of wheat plants with other combinations ofmutations of SBEIIa and SBEIIb of multiple genomes. Nucleotide A.A.Nucleotide A.A. Type Gene Mutation Mutation Gene Mutation MutationSBEIIa Only SBEIIaA G5267A W436* LinkedSBEIIa & IIb SBEIIaB G2386A G233DSBEIIbB C2786T P263L LinkedSBEIIa & IIb SBEIIaD G6306A D433N SBEIIbDC4573T R325W LinkedSBEIIa & IIb SBEIIaA G2605A G264D SBEIIbA G1668AE216K SBEIIa Only SBEIIaB G5038A W436* LinkedSBEIIa & IIb SBEIIaD G6306AD433N SBEIIbD C4573T R325W LinkedSBEIIa & IIb SBEIIaA G2605A G264DSBEIIbA G1668A E216K LinkedSBEIIa & IIb SBEIIaB G2386A G233D SBEIIbBC2786T P263L SBEIIa Only SBEIIaD G6305A W432*

TABLE 12 Additional examples of wheat plants with other combinations ofmutations of SBEIIa and SBEIIb of multiple genomes. Nucleotide A.A.Nucleotide Combo Type Gene Mutation Mutation Gene Mutation A.A. Mutation1 LinkedSBEIIa & IIb SBEIIaA G5267A W436* SBEIIbA G2282A W285*LinkedSBEIIa & IIb SBEIIaB G5038A W436* SBEIIbB G1916A S208NLinkedSBEIIa & IIb SBEIIaD G6305A W432* SBEIIbD G3599A W233* 2 SBEIIaOnly SBEIIaA G5267A W436* SBEIIbA LinkedSBEIIa & IIb SBEIIaB G5038AW436* SBEIIbB G1916A S208N LinkedSBEIIa & IIb SBEIIaD G6305A W432*SBEIIbD G3599A W233* 3 LinkedSBEIIa & IIb SBEIIaA G5267A W436* SBEIIbAG2282A W285* SBEIIa Only SBEIIaB G5038A W436* SBEIIbB LinkedSBEIIa & IIbSBEIIaD G6305A W432* SBEIIbD G3599A W233* 4 LinkedSBEIIa & IIb SBEIIaAG5267A W436* SBEIIbA G2282A W285* LinkedSBEIIa & IIb SBEIIaB G5038AW436* SBEIIbB G1916A S208N SBEIIa Only SBEIIaD G6305A W432* SBEIIbD 5SBEIIa Only SBEIIaA G5267A W436* SBEIIbA SBEIIa Only SBEIIaB G5038AW436* SBEIIbB LinkedSBEIIa & IIb SBEIIaD G6305A W432* SBEIIbD G3599AW233* 6 LinkedSBEIIa & IIb SBEIIaA G5267A W436* SBEIIbA G2282A W285*SBEIIa Only SBEIIaB G5038A W436* SBEIIbB SBEIIa Only SBEIIaD G6305AW432* SBEIIbD 7 SBEIIa Only SBEIIaA G5267A W436* SBEIIbA LinkedSBEIIa &IIb SBEIIaB G5038A W436* SBEIIbB G1916A S208N SBEIIa Only SBEIIaD G6305AW432* SBEIIbD 8 LinkedSBEIIa & IIb SBEIIaA G5267A W436* SBEIIbA G2156ASplice Junction LinkedSBEIIa & IIb SBEIIaB G5038A W436* SBEIIbB C3232TR325W LinkedSBEIIa & IIb SBEIIaD G6305A W432* SBEIIbD C4573T R325W 9SBEIIa Only SBEIIaA G5267A W436* SBEIIbA LinkedSBEIIa & IIb SBEIIaBG5038A W436* SBEIIbB C3232T R325W LinkedSBEIIa & IIb SBEIIaD G6305AW432* SBEIIbD C4573T R325W 10 LinkedSBEIIa & IIb SBEIIaA G5267A W436*SBEIIbA G2156A Splice Junction SBEIIa Only SBEIIaB G5038A W436* SBEIIbBLinkedSBEIIa & IIb SBEIIaD G6305A W432* SBEIIbD C4573T R325W 11LinkedSBEIIa & IIb SBEIIaA G5267A W436* SBEIIbA G2156A Splice JunctionLinkedSBEIIa & IIb SBEIIaB G5038A W436* SBEIIbB C3232T R325W SBEIIa OnlySBEIIaD G6305A W432* SBEIIbD 12 SBEIIa Only SBEIIaA G5267A W436* SBEIIbASBEIIa Only SBEIIaB G5038A W436* SBEIIbB LinkedSBEIIa & IIb SBEIIaDG6305A W432* SBEIIbD C4573T R325W 13 LinkedSBEIIa & IIb SBEIIaA G5267AW436* SBEIIbA G2156A Splice Junction SBEIIa Only SBEIIaB G5038A W436*SBEIIbB SBEIIa Only SBEIIaD G6305A W432* SBEIIbD 14 SBEIIa Only SBEIIaAG5267A W436* SBEIIbA LinkedSBEIIa & IIb SBEIIaB G5038A W436* SBEIIbBC3232T R325W SBEIIa Only SBEIIaD G6305A W432* SBEIIbD 15 LinkedSBEIIa &IIb SBEIIaA G5267A W436* SBEIIbA G2282A W285* LinkedSBEIIa & IIb SBEIIaBG5038A W436* SBEIIbB C3232T R325W LinkedSBEIIa & IIb SBEIIaD G6305AW432* SBEIIbD C4573T R325W 16 LinkedSBEIIa & IIb SBEIIaA G5267A W436*SBEIIbA G2282A W285* LinkedSBEIIa & IIb SBEIIaB G5038A W436* SBEIIbBC3232T R325W LinkedSBEIIa & IIb SBEIIaD G6305A W432* SBEIIbD G3599AW233* 17 LinkedSBEIIa & IIb SBEIIaA G5267A W436* SBEIIbA G2282A W285*LinkedSBEIIa & IIb SBEIIaB G5038A W436* SBEIIbB G1916A S208NLinkedSBEIIa & IIb SBEIIaD G6305A W432* SBEIIbD C4573T R325W 18 SBEIIaOnly SBEIIaA G5267A W436* SBEIIbA LinkedSBEIIa & IIb SBEIIaB G5038AW436* SBEIIbB C3232T R325W LinkedSBEIIa & IIb SBEIIaD G6305A W432*SBEIIbD G3599A W233* 19 SBEIIa Only SBEIIaA G5267A W436* SBEIIbALinkedSBEIIa & IIb SBEIIaB G5038A W436* SBEIIbB G1916A S208NLinkedSBEIIa & IIb SBEIIaD G6305A W432* SBEIIbD G3599A W233* 20 SBEIIaOnly SBEIIaA G5267A W436* SBEIIbA LinkedSBEIIa & IIb SBEIIaB G5038AW436* SBEIIbB G1916A S208N LinkedSBEIIa & IIb SBEIIaD G6305A W432*SBEIIbD C4573T R325W 21 LinkedSBEIIa & IIb SBEIIaA G5267A W436* SBEIIbAG2282A W285* SBEIIa Only SBEIIaB G5038A W436* SBEIIbB LinkedSBEIIa & IIbSBEIIaD G6305A W432* SBEIIbD C4573T R325W 22 LinkedSBEIIa & IIb SBEIIaAG5267A W436* SBEIIbA G2156A Splice Junction SBEIIa Only SBEIIaB G5038AW436* SBEIIbB LinkedSBEIIa & IIb SBEIIaD G6305A W432* SBEIIbD G3599AW233* 23 LinkedSBEIIa & IIb SBEIIaA G5267A W436* SBEIIbA G2282A W285*LinkedSBEIIa & IIb SBEIIaB G5038A W436* SBEIIbB C3232T R325W SBEIIa OnlySBEIIaD G6305A W432* SBEIIbD 24 LinkedSBEIIa & IIb SBEIIaA G5267A W436*SBEIIbA G2156A Splice Junction LinkedSBEIIa & IIb SBEIIaB G5038A W436*SBEIIbB G1916A S208N SBEIIa Only SBEIIaD G6305A W432* SBEIIbD

Mutations in SBEIIa increase amylose content and resistant starch levelsin wheat seeds from (1) double homozygous Kronos wheat plants with astop mutation in SBEIIaA (G5267A/W436*) and a splice junction mutationin SBEIIaB (G5073A/splice junction), and (2) double homozygous Kronoswheat plants with a stop mutation in SBEIIaA (G5267A/W436*) and amissense mutation in SBEIIaB (G5219A/G467E) were evaluated for amylosecontent using the K-AMYL kit from Megazyme (Ireland) and controls ofknown amylose amounts. The amylose content of whole seed milled starchwas an average of 40-49% for the double homozygous mutant seeds comparedto seeds from their wild type sibling controls whose amylose content was20-25%.

Seeds from (1) triple homozygous Express wheat plants with a stopmutation in SBEIIaA (G5267A/W436*), SBEIIaB (G5038A/W436*), and SBEIIaD(G6305A/W432*), and (2) triple homozygous Express wheat plants with astop mutation in SBEIIaA (G5267A/W436*), SBEIIaB (G5069A/W446*), andSBEIIaD (G6335A/W442*) were evaluated for amylose content using theK-AMYL kit from Megazyme (Ireland) and a controls of known amyloseamounts. The amylose content of whole seed milled starch was 50-60% forthe triple homozygous mutant seeds compared to seeds from their wildtype sibling controls whose amylose content was 20-25%.

Flour milled from the triple homozygous mutant seed had 12-15% resistantstarch content compared to flour from the wild type sibling controls,which had approximately 1% resistant starch. Bread made from the triplehomozygous mutant flour had increased resistant starch levels of 6%compared to bread made from flour of wild type sibling and parentalcontrols, which had less than 1% resistant starch. Bread made from a50:50 blend with standard wheat flour had increased resistant starchlevels of 4% compared to bread made from a 50:50 blend with siblingcontrol flour that had resistant starch levels less than 1%.

Seeds from (1) quadruple homozygous Express wheat plants with a linkedmutation in SBEIIaA (G5463A/G472E)—and SBEIIbA (G2282A/W285*), combinedwith a stop mutation in SBEIIaB (G5038A/W436*), and SBEIIaD(G6305A/W432) was evaluated for amylose content using the K-AMYL kitfrom Megazyme (Ireland) and controls of known amylose amounts. Theamylose content of whole seed milled starch was 58% for the quadruplehomozygous mutant seeds compared to seeds from their wild type siblingcontrols whose amylose content was 20-25%.

Seeds from (2) quadruple homozygous Express wheat plants with a stopmutation in SBEIIaA (G5267A/W436*), combined with a stop mutation inSBEIIaB (G5038A/W436*), and a linked mutation in SBEIIaD(G6306A/D433N)—and SBEIIbD (C4573T/R325W) was evaluated for amylosecontent using the K-AMYL kit from Megazyme (Ireland) and controls ofknown amylose amounts. The amylose content of whole seed milled starchwas 38% for the quadruple homozygous mutant seeds compared to seeds fromtheir wild type sibling controls whose amylose content was 23%.

Seeds from (3) quadruple homozygous Express wheat plants with a stopmutation in SBEIIaA (G5267A/W436*), combined with a linked mutation inSBEIIaB (G5219A/G467E)—and SBEIIbB (C3232T/R325W), and a stop mutationin SBEIIaD (G6305A/W432*) were evaluated for amylose content using theK-AMYL kit from Megazyme (Ireland) and controls of known amyloseamounts. The amylose content of whole seed milled starch was 38% for thequadruple homozygous mutant seeds compared to seeds from their wild typesibling controls whose amylose content was 24%.

Seeds from a sextuple homozygous Express wheat plants with linkedmutations in SBEIIaA (G5463A/G472E) and SBEIIbA (G2282A/W285*), combinedwith linked mutations in SBEIIaB (G5219A/G467E) and SBEIIbB(C3232T/R325W), and linked mutations in SBEIIaD (G6306A/D433N) andSBEIIbD (C4573T/R325W) were evaluated for amylose content using theK-AMYL kit from Megazyme (Ireland) and controls of known amyloseamounts. The amylose content of whole seed milled starch was 25-30% forthe sextuple homozygous mutant seeds compared to seeds from their wildtype sibling controls whose amylose content was 16%.

The above examples are provided to illustrate the invention but notlimit its scope. Other variants of the invention will be readilyapparent to one of ordinary skill in the art and are encompassed by theappended claims and all their equivalents. The examples above usedTILLING technology to create and identify mutations in one or more SBEIIgenes of wheat that increase amylose levels in wheat seeds, but one ofordinary skill in the art would understand that other methods such astargeted mutagenesis (also known as site-directed mutagenesis,site-specific mutagenesis or oligonucleotide-directed mutagenesis) couldbe used to create the useful mutations of the present invention in oneor more SBEII loci of wheat (see for example Zhang et al., PNAS107(26):12028-12033, 2010; Saika et al., Plant Physiology 156:1269-1277,2011). All publications, patents, and patent applications cited hereinare hereby incorporated by reference.

1.-18. (canceled)
 19. A process for producing a milled product,comprising the steps of (i) obtaining wheat grain (Triticum aestivum)comprising an embryo and starch, wherein the embryo comprises twoidentical null alleles of an SBEIIa-A gene, two identical null allelesof an SBEIIa-B gene and two identical null alleles of an SBEIIa-D gene,wherein either the two identical null alleles of the SBEIIa-A gene, orof the SBEIIa-B gene or of the SBEIIa-D gene are point mutations,wherein SBEIIa protein is undetectable in the wheat grain, and wherein(a) the starch comprises amylase such that the grain has an amylosecontent of between 50-60% (w/w) as a proportion of the starch in thegrain; and (b) the wheat grain germinates, and (ii) milling the grain,thereby producing the milled product.
 20. The process of claim 19,wherein the amylose content of between 50-60% (w/w) is as measured bythe K-AMYL kit from Megazyme.
 21. The process of claim 19, wherein thewheat grain is free of any exogenous nucleic acid that encodes an RNAwhich reduces expression of an SBEIIa gene.